Chronic infusion of the tryptophan metabolite kynurenine increases mean arterial pressure in male Sprague-Dawley rats.

Chronic kidney disease is the loss of renal function that can occur from aging or through a myriad of other disease states. Rising serum concentrations of kynurenine, a tryptophan metabolite, have been shown to correlate with increasing severity of chronic kidney disease. This study used chronic intravenous infusion in conscious male Sprague-Dawley rats to test the hypothesis that kynurenine can induce renal damage and promote alterations in blood pressure, heart rate, and decreased renal function. We found that kynurenine infusion increased mean arterial pressure, increased the maximum and minimum range of heart rate, decreased glomerular filtration rate, and induced kidney damage in a dose-dependent manner. This study shows that kynurenine infusion can promote kidney disease in healthy, young rats, implying that the increase in kynurenine levels associated with chronic kidney disease may establish a feed-forward mechanism that exacerbates the loss of renal function.NEW & NOTEWORTHY In humans, an elevated serum concentration of kynurenine has long been associated with negative outcomes in various disease states as well as in aging. However, it has been unknown whether these increased kynurenine levels are mediating the disorders or simply associated with them. This study shows that chronically infusing kynurenine can contribute to the development of hypertension and kidney impairment. The mechanism of this action remains to be determined in future studies.

A tamoxifen-inducible Cre knock-in mouse for lens-specific gene manipulation.

Mouse models are valuable tools in studying lens biology and biochemistry, and the Cre-loxP system is the most used technology for gene targeting in the lens. However, numerous genes are indispensable in lens development. The conventional knockout method either prevents lens formation or causes simultaneous cataract formation, hindering the studies of their roles in lens structure, growth, metabolism, and cataractogenesis during lens aging. An inducible Cre-loxP mouse line is an excellent way to achieve such a purpose. We established a lens-specific Cre ERT2 knock-in mouse (LCEK), an inducible mouse model for lens-specific gene targeting in a spatiotemporal manner. LCEK mice were created by in-frame infusion of a P2A-CreERT2 at the C-terminus of the last coding exon of the gene alpha A crystallin (Cryaa). LCEK mice express tamoxifen-inducible Cre recombinase uniquely in the lens. Through ROSAmT/mG and two endogenous genes (Gclc and Rbpj) targeting, we found no Cre recombinase leakage in the lens epithelium, but 50-80% leakage was observed in the lens cortex and nucleus. Administration of tamoxifen almost completely abolished target gene expression in both lens epithelium and cortex but only mildly enhanced gene deletion in the lens nucleus. Notably, no overt leakage of Cre activity was detected in developing LCEK lens when bred with mice carrying loxP floxed genes that are essential for lens development. This newly generated LCEK line will be a powerful tool to target genes in the lens for gene functions study in lens aging, posterior capsule opacification (PCO), and other areas requiring precision gene targeting.

Lipopolysaccharide Pretreatment Prevents Medullary Vascular Congestion following Renal Ischemia by Limiting Early Reperfusion of the Medullary Circulation.

BACKGROUND: Vascular congestion of the renal medulla-trapped red blood cells in the medullary microvasculature-is a hallmark finding at autopsy in patients with ischemic acute tubular necrosis. Despite this, the pathogenesis of vascular congestion is not well defined. METHODS: In this study, to investigate the pathogenesis of vascular congestion and its role in promoting renal injury, we assessed renal vascular congestion and tubular injury after ischemia reperfusion in rats pretreated with low-dose LPS or saline (control). We used laser Doppler flowmetry to determine whether pretreatment with low-dose LPS prevented vascular congestion by altering renal hemodynamics during reperfusion. RESULTS: We found that vascular congestion originated during the ischemic period in the renal venous circulation. In control animals, the return of blood flow was followed by the development of congestion in the capillary plexus of the outer medulla and severe tubular injury early in reperfusion. Laser Doppler flowmetry indicated that blood flow returned rapidly to the medulla, several minutes before recovery of full cortical perfusion. In contrast, LPS pretreatment prevented both the formation of medullary congestion and its associated tubular injury. Laser Doppler flowmetry in LPS-pretreated rats suggested that limiting early reperfusion of the medulla facilitated this protective effect, because it allowed cortical perfusion to recover and clear congestion from the large cortical veins, which also drain the medulla. CONCLUSIONS: Blockage of the renal venous vessels and a mismatch in the timing of cortical and medullary reperfusion results in congestion of the outer medulla's capillary plexus and promotes early tubular injury after renal ischemia. These findings indicate that hemodynamics during reperfusion contribute to the renal medulla's susceptibility to ischemic injury.

Blocking ribosomal protein S6 phosphorylation inhibits podocyte hypertrophy and focal segmental glomerulosclerosis.

Ribosomal protein S6 (rpS6) phosphorylation mediates the hypertrophic growth of kidney proximal tubule cells. However, the role of rpS6 phosphorylation in podocyte hypertrophy and podocyte loss during the pathogenesis of focal segmental glomerulosclerosis (FSGS) remains undefined. Here, we examined rpS6 phosphorylation levels in kidney biopsy specimens from patients with FSGS and in podocytes from mouse kidneys with Adriamycin-induced FSGS. Using genetic and pharmacologic approaches in the mouse model of FSGS, we investigated the role of rpS6 phosphorylation in podocyte hypertrophy and loss during development and progression of FSGS. Phosphorylated rpS6 was found to be markedly increased in the podocytes of patients with FSGS and Adriamycin-induced FSGS mice. Genetic deletion of the Tuberous sclerosis 1 gene in kidney glomerular podocytes activated mammalian target of rapamycin complex 1 signaling to rpS6 phosphorylation, resulting in podocyte hypertrophy and pathologic features similar to those of patients with FSGS including podocyte loss, leading to segmental glomerulosclerosis. Since protein phosphatase 1 is known to negatively regulate rpS6 phosphorylation, treatment with an inhibitor increased phospho-rpS6 levels, promoted podocyte hypertrophy and exacerbated formation of FSGS lesions. Importantly, blocking rpS6 phosphorylation (either by generating congenic rpS6 knock-in mice expressing non-phosphorylatable rpS6 or by inhibiting ribosomal protein S6 kinase 1-mediated rpS6 phosphorylation with an inhibitor) significantly blunted podocyte hypertrophy, inhibited podocyte loss, and attenuated formation of FSGS lesions. Thus, our study provides genetic and pharmacologic evidence indicating that specifically targeting rpS6 phosphorylation can attenuate the development of FSGS lesions by inhibiting podocyte hypertrophy and associated podocyte depletion.

Cre/loxP approach-mediated downregulation of Pik3c3 inhibits the hypertrophic growth of renal proximal tubule cells.

Nephron loss stimulates residual functioning nephrons to undergo compensatory growth. Excessive nephron growth may be a maladaptive response that sets the stage for progressive nephron damage, leading to kidney failure. To date, however, the mechanism of nephron growth remains incompletely understood. Our previous study revealed that class III phosphatidylinositol-3-kinase (Pik3c3) is activated in the remaining kidney after unilateral nephrectomy (UNX)-induced nephron loss, but previous studies failed to generate a Pik3c3 gene knockout animal model. Global Pik3c3 deletion results in embryonic lethality. Given that renal proximal tubule cells make up the bulk of the kidney and undergo the most prominent hypertrophic growth after UNX, in this study we used Cre-loxP-based approaches to demonstrate for the first time that tamoxifen-inducible SLC34a1 promoter-driven CreERT2 recombinase-mediated downregulation of Pik3c3 expression in renal proximal tubule cells alone is sufficient to inhibit UNX- or amino acid-induced hypertrophic nephron growth. Furthermore, our mechanistic studies unveiled that the SLC34a1-CreERT2 recombinase-mediated Pik3c3 downregulation inhibited UNX- or amino acid-stimulated lysosomal localization and signaling activation of mechanistic target of rapamycin complex 1 (mTORC1) in the renal proximal tubules. Moreover, our additional cell culture experiments using RNAi confirmed that knocking down Pik3c3 expression inhibited amino acid-stimulated mTORC1 signaling and blunted cellular growth in primary cultures of renal proximal tubule cells. Together, both our in vivo and in vitro experimental results indicate that Pik3c3 is a major mechanistic mediator responsible for sensing amino acid availability and initiating hypertrophic growth of renal proximal tubule cells by activation of the mTORC1-S6K1-rpS6 signaling pathway.

The expression level of class III phosphatidylinositol-3 kinase controls the degree of compensatory nephron hypertrophy.

Excessive compensatory nephron hypertrophy (CNH) has been implicated in setting the stage for progressive nephron damage. Lack of a class III phosphatidylinositol 3-kinase (Pik3c3) inhibitor suitable for using in animals and lack of a Pik3c3-deficient animal model preclude the possibility of conclusively defining a role for Pik3c3 in CNH in previous studies. Here, we report that insertion of an Frt-flanked PGK-Neo cassette into intron 19 of the mouse Pik3c3 gene resulted in a hypomorphic allele. This allowed us to create a unique mouse model and provide the first definitive genetic evidence demonstrating whether Pik3c3 is essential for the regulation of CNH. Our results indicate that homozygous Pik3c3 hypomorphic (Pik3c3Hypo/Hypo) mice express significantly low levels of Pik3c3 than heterozygous Pik3c3 hypomorphic (Pik3c3Hypo/WT) littermates, which already express a lower level of Pik3c3 than wild-type (Pik3c3WT/WT) littermates. Interestingly, after unilateral nephrectomy (UNX), Pik3c3Hypo/Hypo mice develop a significantly lower degree of CNH than Pik3c3WT/WT mice and Pik3c3Hypo/WT mice, as revealed by measurement of kidney weight, kidney-to-body weight ratio, renal protein-to-DNA ratio, and morphometric analysis of proximal tubular and glomerular size. Mechanistically, UNX-induced mammalian target of rapamycin complex 1 (mTORC1) signaling to phosphorylation of ribosomal protein S6 (rpS6) in the remaining kidney was markedly inhibited in Pik3c3 hypomorphic mice. In conclusion, the present study reports a Pik3c3 hypomorphic mouse model and provides the first definitive evidence that Pik3c3 controls the degree of compensatory nephron hypertrophy. In addition, our signaling data provide the first definitive in vivo proof that Pik3c3 functions upstream of the mTORC1-S6 kinase 1-rpS6 pathway in the regulation of compensatory nephron hypertrophy.

The TNF-derived TIP peptide activates the epithelial sodium channel and ameliorates experimental nephrotoxic serum nephritis.

In mice, the initial stage of nephrotoxic serum-induced nephritis (NTN) mimics antibody-mediated human glomerulonephritis. Local immune deposits generate tumor necrosis factor (TNF), which activates pro-inflammatory pathways in glomerular endothelial cells (GECs) and podocytes. Because TNF receptors mediate antibacterial defense, existing anti-TNF therapies can promote infection; however, we have previously demonstrated that different functional domains of TNF may have opposing effects. The TIP peptide mimics the lectin-like domain of TNF, and has been shown to blunt inflammation in acute lung injury without impairing TNF receptor-mediated antibacterial activity. We evaluated the impact of TIP peptide in NTN. Intraperitoneal administration of TIP peptide reduced inflammation, proteinuria, and blood urea nitrogen. The protective effect was blocked by the cyclooxygenase inhibitor indomethacin, indicating involvement of prostaglandins. Targeted glomerular delivery of TIP peptide improved pathology in moderate NTN and reduced mortality in severe NTN, indicating a local protective effect. We show that TIP peptide activates the epithelial sodium channel(ENaC), which is expressed by GEC, upon binding to the channel's α subunit. In vitro, TNF treatment of GEC activated pro-inflammatory pathways and decreased the generation of prostaglandin E2 and nitric oxide, which promote recovery from NTN. TIP peptide counteracted these effects. Despite the capacity of TIP peptide to activate ENaC, it did not increase mean arterial blood pressure in mice. In the later autologous phase of NTN, TIP peptide blunted the infiltration of Th17 cells. By countering the deleterious effects of TNF through direct actions in GEC, TIP peptide could provide a novel strategy to treat glomerular inflammation.

Autophagy in diabetic kidney disease: regulation, pathological role and therapeutic potential.

Diabetic kidney disease, a leading cause of end-stage renal disease, has become a serious public health problem worldwide and lacks effective therapies. Autophagy is a highly conserved lysosomal degradation pathway that removes protein aggregates and damaged organelles to maintain cellular homeostasis. As important stress-responsive machinery, autophagy is involved in the pathogenesis of various diseases. Emerging evidence has suggested that dysregulated autophagy may contribute to both glomerular and tubulointerstitial pathologies in kidneys under diabetic conditions. This review summarizes the recent findings regarding the role of autophagy in the pathogenesis of diabetic kidney disease and highlights the regulation of autophagy by the nutrient-sensing pathways and intracellular stress signaling in this disease. The advances in our understanding of autophagy in diabetic kidney disease will facilitate the discovery of a new therapeutic target for the prevention and treatment of this life-threatening diabetes complication.

Dicer deficiency in proximal tubules exacerbates renal injury and tubulointerstitial fibrosis and upregulates Smad2/3.

Renal fibrosis is a common pathological feature in chronic kidney disease (CKD), including diabetic kidney disease (DKD) and obstructive nephropathy. Multiple microRNAs have been implicated in the pathogenesis of both DKD and obstructive nephropathy, although the overall role of microRNAs in tubular injury and renal fibrosis in CKD is unclear. Dicer (a key RNase III enzyme for microRNA biogenesis) was specifically ablated from kidney proximal tubules in mice via the Cre-lox system to deplete micoRNAs. Proximal tubular Dicer knockout (PT- Dicer KO) mice and wild-type (WT) littermates were subjected to streptozotocin (STZ) treatment to induce DKD or unilateral ureteral obstruction (UUO) to induce obstructive nephropathy. Renal hypertrophy, renal tubular apoptosis, kidney inflammation, and tubulointerstitial fibrosis were examined. Compared with WT mice, PT- Dicer KO mice showed more severe tubular injury and renal inflammation following STZ treatment. These mice also developed higher levels of tubolointerstitial fibrosis. Meanwhile, PT- Dicer KO mice had a significantly higher Smad2/3 expression in kidneys than WT mice (at 6 mo of age) in both control and STZ-treated mice. Similarly, UUO induced more severe renal injury, inflammation, and interstitial fibrosis in PT- Dicer KO mice than WT. Although we did not detect obvious Smad2/3 expression in sham-operated mice (2-3 mo old), significantly more Smad2/3 was induced in obstructed PT- Dicer KO kidneys. These results supported a protective role of Dicer-dependent microRNA synthesis in renal injury and fibrosis development in CKD, specifically in DKD and obstructive nephropathy. Depletion of Dicer and microRNAs may upregulate Smad2/3-related signaling pathway to enhance the progression of CKD.

Chronic renal artery insulin infusion increases mean arterial pressure in male Sprague-Dawley rats.

Hyperinsulinemia has been hypothesized to cause hypertension in obesity, type 2 diabetes, and metabolic syndrome through a renal mechanism. However, it has been challenging to isolate renal mechanisms in chronic experimental models due, in part, to technical difficulties. In this study, we tested the hypothesis that a renal mechanism underlies insulin hypertension. We developed a novel technique to permit continuous insulin infusion through the renal artery in conscious rats for 7 days. Mean arterial pressure increased by ~10 mmHg in rats that were infused intravenously (IV) with insulin and glucose. Renal artery doses were 20% of the intravenous doses and did not raise systemic insulin levels or cause differences in blood glucose. The increase in blood pressure was not different from the IV group. Mean arterial pressure did not change in vehicle-infused rats, and there were no differences in renal injury scoring due to the renal artery catheter. Glomerular filtration rate, plasma renin activity, and urinary sodium excretion did not differ between groups at baseline and did not change significantly with insulin infusion. Thus, by developing a novel approach for chronic, continuous renal artery insulin infusion, we provided new evidence that insulin causes hypertension in rats through actions initiated within the kidney.

Protein Kinase Cδ Suppresses Autophagy to Induce Kidney Cell Apoptosis in Cisplatin Nephrotoxicity.

Nephrotoxicity is a major adverse effect in cisplatin chemotherapy, and renoprotective approaches are unavailable. Recent work unveiled a critical role of protein kinase Cδ (PKCδ) in cisplatin nephrotoxicity and further demonstrated that inhibition of PKCδ not only protects kidneys but enhances the chemotherapeutic effect of cisplatin in tumors; however, the underlying mechanisms remain elusive. Here, we show that cisplatin induced rapid activation of autophagy in cultured kidney tubular cells and in the kidneys of injected mice. Cisplatin also induced the phosphorylation of mammalian target of rapamycin (mTOR), p70S6 kinase downstream of mTOR, and serine/threonine-protein kinase ULK1, a component of the autophagy initiating complex. In vitro, pharmacologic inhibition of mTOR, directly or through inhibition of AKT, enhanced autophagy after cisplatin treatment. Notably, in both cells and kidneys, blockade of PKCδ suppressed the cisplatin-induced phosphorylation of AKT, mTOR, p70S6 kinase, and ULK1 resulting in upregulation of autophagy. Furthermore, constitutively active and inactive forms of PKCδ respectively enhanced and suppressed cisplatin-induced apoptosis in cultured cells. In mechanistic studies, we showed coimmunoprecipitation of PKCδ and AKT from lysates of cisplatin-treated cells and direct phosphorylation of AKT at serine-473 by PKCδin vitro Finally, administration of the PKCδ inhibitor rottlerin with cisplatin protected against cisplatin nephrotoxicity in wild-type mice, but not in renal autophagy-deficient mice. Together, these results reveal a pathway consisting of PKCδ, AKT, mTOR, and ULK1 that inhibits autophagy in cisplatin nephrotoxicity. PKCδ mediates cisplatin nephrotoxicity at least in part by suppressing autophagy, and accordingly, PKCδ inhibition protects kidneys by upregulating autophagy.

Exosome production and its regulation of EGFR during wound healing in renal tubular cells.

Kidney repair following injury involves the reconstitution of a structurally and functionally intact tubular epithelium. Growth factors and their receptors, such as EGFR, are important in the repair of renal tubules. Exosomes are cell-produced small (~100 nm in diameter) vesicles that contain and transfer proteins, lipids, RNAs, and DNAs between cells. In this study, we examined the relationship between exosome production and EGFR activation and the potential role of exosome in wound healing. EGFR activation occurred shortly after scratch wounding in renal tubular cells. Wound repair after scratching was significantly promoted by EGF and suppressed by EGFR inhibitor gefitinib. Interestingly, scratch wounding induced a significant increase of exosome production. The exosome production was decreased by EGF and increased by gefitinib, suggesting a suppressive role of EGFR signaling in exosome production. Conversely, inhibition of exosome release by GW4869 and manumycin A markedly increased EGFR activation and promoted wound healing. Moreover, exosomes derived from scratch-wounding cells could inhibit wound healing. Collectively, the results indicate that wound healing in renal tubular cells is associated with EGFR activation and exosome production. Although EGFR activation promotes wound healing, released exosomes may antagonize EGFR activation and wound healing.

Autophagy is activated to protect against podocyte injury in adriamycin-induced nephropathy.

Podocytes are highly differentiated epithelial cells wrapping glomerular capillaries to form the filtration barrier in kidneys. As such, podocyte injury or dysfunction is a critical pathogenic event in glomerular disease. Autophagy plays an important role in the maintenance of the homeostasis and function of podocytes. However, it is less clear whether and how autophagy contributes to podocyte injury in glomerular disease. Here, we have examined the role of autophagy in adriamycin-induced nephropathy, a classic model of glomerular disease. We show that autophagy was induced by adriamycin in cultured podocytes in vitro and in podocytes in mice. In cultured podocytes, activation of autophagy with rapamycin led to the suppression of adriamycin-induced apoptosis, whereas inhibition of autophagy with chloroquine enhanced podocyte apoptosis during adriamycin treatment. To determine the role of autophagy in vivo, we established an inducible podocyte-specific autophagy-related gene 7 knockout mouse model (Podo-Atg7-KO). Compared with wild-type littermates, Podo-Atg7-KO mice showed higher levels of podocyte injury, glomerulopathy, and proteinuria during adriamycin treatment. Together, these observations support an important role of autophagy in protecting podocytes under the pathological conditions of glomerular disease, suggesting the therapeutic potential of autophagy induction.

DNA methylation protects against cisplatin-induced kidney injury by regulating specific genes, including interferon regulatory factor 8.

DNA methylation is an epigenetic mechanism that regulates gene transcription without changing primary nucleotide sequences. In mammals, DNA methylation involves the covalent addition of a methyl group to the 5-carbon position of cytosine by DNA methyltransferases (DNMTs). The change of DNA methylation and its pathological role in acute kidney injury (AKI) remain largely unknown. Here, we analyzed genome-wide DNA methylation during cisplatin-induced AKI by reduced representation bisulfite sequencing. This technique identified 215 differentially methylated regions between the kidneys of control and cisplatin-treated animals. While most of the differentially methylated regions were in the intergenic, intronic, and coding DNA sequences, some were located in the promoter or promoter-regulatory regions of 15 protein-coding genes. To determine the pathological role of DNA methylation, we initially examined the effects of the DNA methylation inhibitor 5-aza-2'-deoxycytidine and showed it increased cisplatin-induced apoptosis in a rat kidney proximal tubular cell line. We further established a kidney proximal tubule-specific DNMT1 (PT-DNMT1) knockout mouse model, which showed more severe AKI during cisplatin treatment than wild-type mice. Finally, interferon regulatory factor 8 (Irf8), a pro-apoptotic factor, was identified as a hypomethylated gene in cisplatin-induced AKI, and this hypomethylation was associated with a marked induction of Irf8. In the rat kidney proximal tubular cells, the knockdown of Irf8 suppressed cisplatin-induced apoptosis, supporting a pro-death role of Irf8 in renal tubular cells. Thus, DNA methylation plays a protective role in cisplatin-induced AKI by regulating specific genes, such as Irf8.

Blocking rpS6 Phosphorylation Exacerbates Tsc1 Deletion-Induced Kidney Growth.

The molecular mechanisms underlying renal growth and renal growth-induced nephron damage remain poorly understood. Here, we report that in murine models, deletion of the tuberous sclerosis complex protein 1 (Tsc1) in renal proximal tubules induced strikingly enlarged kidneys, with minimal cystogenesis and occasional microscopic tumorigenesis. Signaling studies revealed hyperphosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and increased phosphorylation of ribosomal protein S6 (rpS6) in activated renal tubules. Notably, knockin of a nonphosphorylatable rpS6 in these Tsc1-mutant mice exacerbated cystogenesis and caused drastic nephron damage and renal fibrosis, leading to kidney failure and a premature death rate of 67% by 9 weeks of age. In contrast, Tsc1 single-mutant mice were all alive and had far fewer renal cysts at this age. Mechanistic studies revealed persistent activation of mammalian target of rapamycin complex 1 (mTORC1) signaling causing hyperphosphorylation and consequent accumulation of 4E-BP1, along with greater cell proliferation, in the renal tubules of Tsc1 and rpS6 double-mutant mice. Furthermore, pharmacologic treatment of Tsc1 single-mutant mice with rapamycin reduced hyperphosphorylation and accumulation of 4E-BP1 but also inhibited phosphorylation of rpS6. Rapamycin also exacerbated cystic and fibrotic lesions and impaired kidney function in these mice, consequently leading to a premature death rate of 40% within 2 weeks of treatment, despite destroying tumors and decreasing kidney size. These findings indicate that Tsc1 prevents aberrant renal growth and tumorigenesis by inhibiting mTORC1 signaling, whereas phosphorylated rpS6 suppresses cystogenesis and fibrosis in Tsc1-deleted kidneys.

MicroRNA-489 Induction by Hypoxia-Inducible Factor-1 Protects against Ischemic Kidney Injury.

MicroRNAs have been implicated in ischemic AKI. However, the specific microRNA species that regulates ischemic kidney injury remains unidentified. Our previous microarray analysis revealed microRNA-489 induction in kidneys of mice subjected to renal ischemia-reperfusion. In this study, we verified the induction of microRNA-489 during ischemic AKI in mice and further examined the underlying mechanisms. Hypoxia-inducible factor-1α deficiency associated with diminished microRNA-489 induction in cultured rat proximal tubular cells subjected to hypoxia and kidney tissues of mice after renal ischemia-reperfusion injury. Moreover, genomic analysis revealed that microRNA-489 is intronic in the calcitonin receptor gene, and chromatin immunoprecipitation assays showed increased binding of hypoxia-inducible factor-1 to a specific site in the calcitonin receptor gene promoter after hypoxia. Inhibition of microRNA-489 increased apoptosis in renal tubular cells after ATP depletion injury in vitro, whereas microRNA-489 mimics mediated protection. In mice, inhibition of microRNA-489 enhanced tubular cell death and ischemic AKI without significantly affecting tubular cell proliferation. Deep sequencing identified 417 mRNAs that were recruited to the RNA-induced silencing complex by microRNA-489. Of the identified mRNAs, 127 contain microRNA-489 targeting sites, and of those, 18 are involved in the cellular stress response, including the poly(ADP-ribose) polymerase 1 gene implicated in ischemic kidney injury. Sequence analysis and in vitro studies validated poly(ADP-ribose) polymerase 1 as a microRNA-489 target. Together, these results suggest that microRNA-489 is induced via hypoxia-inducible factor-1 during ischemic AKI to protect kidneys by targeting relevant genes.

Enzymatic hydrolysis of Grass Carp fish skin hydrolysates able to promote the proliferation of Streptococcus thermophilus.

BACKGROUND: The promotion effect on proliferation of Streptococcus thermophilus by enzymatic hydrolysates of aquatic products was firstly studied. The effect of influencing factors of the hydrolysis on the growth of S. thermophilus was investigated. RESULT: Grass Carp fish skin was hydrolysed to peptides by enzymatic hydrolysis using protease ProteAX, and for the S. thermophilus growth, the optimal enzymatic hydrolysis conditions were temperature of 60 °C, initial pH of 9.0, enzyme concentration of 10 g kg-1 , hydrolysis time of 80 min, and ratio of material to liquid of 1:2. The Grass Carp fish skin hydrolysate (GCFSH) prepared under the optimum conditions was fractionated to five fragments (GCFSH 1, GCFSH 2, GCFSH 3, GCFSH 4, GCFSH 5) according to molecular weight sizes, in which the fragments GCFSH 4 and GCFSH 5, with molecular weights of less than 1000 Da, significantly promoted the growth of S. thermophilus. CONCLUSION: The hydrolysis process of Grass Carp fish skin can be simplified, and the peptides with molecular weights below 1000 Da in the hydrolysates are the best nitrogen source for proliferation of S. thermophilus. This work can provide a fundamental theoretical basis for the production of multi-component functional foods, especially in milk drinks or yogurt. © 2017 Society of Chemical Industry.

EGF receptor deletion in podocytes attenuates diabetic nephropathy.

The generation of reactive oxygen species (ROS), particularly superoxide, by damaged or dysfunctional mitochondria has been postulated to be an initiating event in the development of diabetes complications. The glomerulus is a primary site of diabetic injury, and podocyte injury is a classic hallmark of diabetic glomerular lesions. In streptozotocin-induced type 1 diabetes, podocyte-specific EGF receptor (EGFR) knockout mice (EGFR(podKO)) and their wild-type (WT) littermates had similar levels of hyperglycemia and polyuria, but EGFR(podKO) mice had significantly less albuminuria and less podocyte loss compared with WT diabetic mice. Furthermore, EGFR(podKO) diabetic mice had less TGF-ß1 expression, Smad2/3 phosphorylation, and glomerular fibronectin deposition. Immunoblotting of isolated glomerular lysates revealed that the upregulation of cleaved caspase 3 and downregulation of Bcl2 in WT diabetic mice were attenuated in EGFR(podKO) diabetic mice. Administration of the SOD mimetic mito-tempol or the NADPH oxidase inhibitor apocynin attenuated the upregulation of p-c-Src, p-EGFR, p-ERK1/2, p-Smad2/3, and TGF-ß1 expression and prevented the alteration of cleaved caspase 3 and Bcl2 expression in glomeruli of WT diabetic mice. High-glucose treatment of cultured mouse podocytes induced similar alterations in the production of ROS; phosphorylation of c-Src, EGFR, and Smad2/3; and expression of TGF-ß1, cleaved caspase 3, and Bcl2. These alterations were inhibited by treatment with mito-tempol or apocynin or by inhibiting EGFR expression or activity. Thus, results of our studies utilizing mice with podocyte-specific EGFR deletion demonstrate that EGFR activation has a major role in activating pathways that mediate podocyte injury and loss in diabetic nephropathy.

Phosphorylation of ribosomal protein S6 mediates compensatory renal hypertrophy.

The molecular mechanism underlying renal hypertrophy and progressive nephron damage remains poorly understood. Here we generated congenic ribosomal protein S6 (rpS6) knock-in mice expressing nonphosphorylatable rpS6 and found that uninephrectomy-induced renal hypertrophy was significantly blunted in these knock-in mice. Uninephrectomy-induced increases in cyclin D1 and decreases in cyclin E in the remaining kidney were attenuated in the knock-in mice compared with their wild-type littermates. Uninephrectomy induced rpS6 phosphorylation in the wild-type mice; however, no rpS6 phosphorylation was detected in uninephrectomized or sham-operated knock-in mice. Nonetheless, uninephrectomy stimulated comparable 4E-BP1 phosphorylation in both knock-in and wild-type mice, indicating that mTORC1 was still activated in the knock-in mice. Moreover, the mTORC1 inhibitor rapamycin prevented both rpS6 and 4E-BP1 phosphorylation, significantly blunted uninephrectomy-induced renal hypertrophy in wild-type mice, but did not prevent residual renal hypertrophy despite inhibiting 4E-BP1 phosphorylation in uninephrectomized knock-in mice. Thus, both genetic and pharmacological approaches unequivocally demonstrate that phosphorylated rpS6 is a downstream effector of the mTORC1-S6K1 signaling pathway mediating renal hypertrophy. Hence, rpS6 phosphorylation facilitates the increase in cyclin D1 and decrease in cyclin E1 that underlie the hypertrophic nature of uninephrectomy-induced kidney growth.

Hyperglycemia, p53, and mitochondrial pathway of apoptosis are involved in the susceptibility of diabetic models to ischemic acute kidney injury.

Patients with chronic kidney diseases, including diabetic nephropathy, are more susceptible to acute kidney injury (AKI) and have a worse prognosis following AKI. However, the underlying mechanism is unclear. Here we tested whether diabetic mice were more sensitive to AKI and show that renal ischemia-reperfusion induced significantly more severe AKI and higher mortality in the streptozotocin and Akita diabetic mouse models. The severity of AKI in the mice correlated with their blood glucose levels. In vitro, high glucose-conditioned renal proximal tubular cells showed higher apoptosis and caspase activation following ATP depletion and hypoxic injury, accompanied by a heightened mitochondrial accumulation of Bax and release of cytochrome c. In response to injury, both glucose-conditioned renal proximal tubular cells and diabetic kidney tissues showed markedly higher p53 induction. Suppression of p53 diminished the sensitivity of high glucose-conditioned cells to acute injury in vitro. Moreover, blockade of p53 by pifithrin-α, siRNA, or proximal tubule-targeted gene ablation reduced ischemic AKI in diabetic mice. Insulin reduced blood glucose in diabetic mice and largely attenuated their AKI sensitivity. Thus, our results suggest the involvement of hyperglycemia, p53, and the mitochondrial pathway of apoptosis in the susceptibility of diabetic models to AKI.