Demonstration of constant nitrogen and energy amounts in pig urine under acidic conditions at room temperature and determination of the minimum amount of hydrochloric acid required for nitrogen preservation in pig urine.

OBJECTIVE: The objectives were to demonstrate that the nitrogen and energy in pig urine supplemented with hydrochloric acid (HCl) are not volatilized and to determine the minimum amount of HCl required for nitrogen preservation from pig urine. METHODS: In Exp. 1, urine samples of 3.0 L each with 5 different nitrogen concentrations were divided into 2 groups: 1.5 L of urine added with i) 100 mL of distilled water or ii) 100 mL of 6 N HCl. The urine in open plastic containers was placed on a laboratory table at room temperature for 10 d. The weight, nitrogen concentration, and gross energy concentration of the urine samples were determined every 2 d. In Exp. 2, three urine samples with different nitrogen concentrations were added with different amounts of 6 N HCl to obtain varying pH values. All urine samples were placed on a laboratory table for 5 d followed by nitrogen analysis. RESULTS: Nitrogen amounts in urine supplemented with distilled water decreased linearly with time, whereas those supplemented with 6 N HCl remained constant. Based on the linear broken-line analysis, nitrogen was not volatilized at a pH below 5.12 (standard error = 0.71 and p<0.01). In Exp. 3, an equation for determining the amount of 6 N HCl to preserve nitrogen in pig urine was developed: additional 6 N HCl (mL) to 100 mL of urine = 3.83×nitrogen in urine (g/100 mL)+0.71 with R2 = 0.96 and p<0.01. If 62.7 g/d of nitrogen is excreted, at least 240 mL of 6 N HCl should be added to the urine collection container. CONCLUSION: Nitrogen in pig urine is not volatilized at a pH below 5.12 at room temperature and the amount of 6 N HCl required for nitrogen preservation may be up to 240 mL per day for a 110-kg pig depending on urinary nitrogen excretion.

Mouse Metanephric Mesenchymal Cell-Derived Angioblasts Undergo Vasculogenesis in Three-Dimensional Culture.

In vitro models for the investigation of renal vascular development are limited. We previously showed that isolated metanephric mesenchymal (MM) and ureteric bud (UB) cells grown in three-dimensional (3D) matrices formed organoids that consisted of primitive vascular structures surrounding a polarized epithelium. Here, we examined the potential of two principal effectors of vasculogenesis, vascular endothelial growth factor A (VEGF-A), and platelet-derived growth factor B chain (PDGF-BB), to stimulate MM cell differentiation. The results showed that MM cells possess angioblast characteristics by expressing phenotypic markers for endothelial and mesenchymal cells. UB cells synthesize VEGF-A and PDGF-BB proteins and RNA, whereas the MM cells express the respective cognate receptors, supporting their role in directional induction of vasculogenesis. VEGF-A stimulated proliferation of MM cells in monolayer and in 3D sponges but did not affect MM cell migration, organization, or vasculogenesis. However, PDGF-BB stimulated MM cell proliferation, migration, and vasculogenesis in monolayer and organization of the cells into primitive capillary-like assemblies in 3D sea sponge scaffolds in vitro. A role for PDGF-BB in vasculogenesis in the 3D MM/UB co-culture system was validated by direct interference with PDGF-BB or PDGF receptor-ß cell interactions to implicate PDGF-BB as a primary effector of MM cell vasculogenesis. Thus, MM cells resemble early renal angioblasts that may provide an ideal platform for the investigation of renal vasculogenesis in vitro.

Molecular events in matrix protein metabolism in the aging kidney.

We explored molecular events associated with aging-induced matrix changes in the kidney. C57BL6 mice were studied in youth, middle age, and old age. Albuminuria and serum cystatin C level (an index of glomerular filtration) increased with aging. Renal hypertrophy was evident in middle-aged and old mice and was associated with glomerulomegaly and increase in mesangial fraction occupied by extracellular matrix. Content of collagen types I and III and fibronectin was increased with aging; increment in their mRNA varied with the phase of aging. The content of ZEB1 and ZEB2, collagen type I transcription inhibitors, and their binding to the collagen type Iα2 promoter by ChIP assay also showed age-phase-specific changes. Lack of increase in mRNA and data from polysome assay suggested decreased degradation as a potential mechanism for kidney collagen type I accumulation in the middle-aged mice. These changes occurred with increment in TGFß mRNA and protein and activation of its SMAD3 pathway; SMAD3 binding to the collagen type Iα2 promoter was also increased. TGFß-regulated microRNAs (miRs) exhibited selective regulation. The renal cortical content of miR-21 and miR-200c, but not miR-192, miR-200a, or miR-200b, was increased with aging. Increased miR-21 and miR-200c contents were associated with reduced expression of their targets, Sprouty-1 and ZEB2, respectively. These data show that aging is associated with complex molecular events in the kidney that are already evident in the middle age and progress to old age. Age-phase-specific regulation of matrix protein synthesis occurs and involves matrix protein-specific transcriptional and post-transcriptional mechanisms.

Reciprocal induction of simple organogenesis by mouse kidney progenitor cells in three-dimensional co-culture.

Kidney development is regulated by a coordinated reciprocal induction of metanephric mesenchymal (MM) and ureteric bud (UB) cells. Here, established MM and UB progenitor cell lines were recombined in three-dimensional Matrigel implants in SCID mice. Differentiation potential was examined for changes in phenotype, organization, and the presence of specialized proteins using immunofluorescence and bright-field and electron microscopy. Both cell types, when grown alone, did not develop into specialized structures. When combined, the cells organized into simple organoid structures of polarized epithelia with lumens surrounded by capillary-like structures. Tracker experiments indicated the UB cells formed the tubuloid structures, and the MM cells were the source of the capillary-like cells. The epithelial cells stained positive for pancytokeratin, the junctional complex protein ZO-1, collagen type IV, as well as UB and collecting duct markers, rearranged during transfection (RET), Dolichos biflorus lectin, EndoA cytokeratin, and aquaporin 2. The surrounding cells expressed α-smooth muscle actin, vimentin, platelet endothelial cell adhesion molecule 1 (PECAM), and aquaporin 1, a marker of vasculogenesis. The epithelium exhibited apical vacuoles, microvilli, junctional complexes, and linear basement membranes. Capillary-like structures showed endothelial features with occasional pericytes. UB cell epithelialization was augmented in the presence of MM cell-derived conditioned medium, glial-derived neurotrophic factor (GDNF), hepatocyte growth factor (HGF), or fibronectin. MM cells grown in the presence of UB-derived conditioned medium failed to undergo differentiation. However, UB cell-derived conditioned medium induced MM cell migration. These studies indicate that tubulogenesis and vasculogenesis can be partially recapitulated by recombining individual MM and UB cell lineages, providing a new model system to study organogenesis ex vivo.

Ribosomal biogenesis induction by high glucose requires activation of upstream binding factor in kidney glomerular epithelial cells.

Diabetes promotes protein synthesis to induce kidney hypertrophy and increase renal matrix proteins. Increased capacity for mRNA translation by way of ribosomal biogenesis facilitates sustained stimulation of protein synthesis. We tested the hypothesis that high glucose induces ribosomal biogenesis as indicated by an increase in rRNA synthesis in the setting of augmented protein synthesis. High glucose (30 mM) increased global protein synthesis, expression of matrix proteins, laminin γ1 and fibronectin, and rDNA transcription in glomerular epithelial cells (GECs) compared with 5 mM glucose. High glucose induced Ser388 phosphorylation of upstream binding factor (UBF), an rDNA transcription factor, along with increased phosphorylation of Erk and p70S6 kinase. Inactivation of Erk and p70S6 kinase either by their respective chemical inhibitors or by expression of their inactive mutant constructs blocked high-glucose-induced UBF phosphorylation. High glucose reduced nuclear content of p19ARF and promoted dissolution of inactive UBF-p19ARF complex. High glucose also promoted association of UBF with RPA194, a subunit of RNA polymerase I. Inhibition of Erk, p70S6 kinase, and UBF1 by transfecting GECs with their respective inactive mutants abolished laminin γ1 synthesis, protein synthesis, and rDNA transcription. Renal cortex from type 1 diabetic rats and type 2 diabetic db/db mice showed increased phosphorylation of UBF, Erk, and p70S6 kinase coinciding with renal hypertrophy and onset of matrix accumulation. Our data suggest that augmented ribosome biogenesis occurs in an UBF-dependent manner during increased protein synthesis induced by high glucose in the GECs that correlates with UBF activation and renal hypertrophy in rodents with type 1 and type 2 diabetes.

Acute hyperglycemia rapidly stimulates VEGF mRNA translation in the kidney. Role of angiotensin type 2 receptor (AT2).

Angiotensin II (Ang II) and vascular endothelial growth factor (VEGF) are important mediators of kidney injury in diabetes. Acute hyperglycemia increased synthesis of intrarenal Ang I and Ang II and resulted in activation of both Ang II receptors, AT1 and AT2, in the kidney. Losartan (specific AT1 antagonist) or PD123319 (specific AT2 antagonist) did not affect hyperglycemia but prevented activation of renal AT1 and AT2, respectively. In murine renal cortex, acute hyperglycemia increased VEGF protein but not mRNA content after 24 h, which suggested translational regulation. Blockade of AT2, but not AT1, prevented increase in VEGF synthesis by inhibiting translation of VEGF mRNA in renal cortex. Acute hyperglycemia increased VEGF expression in wild type but not in AT2 knockout mice. Binding of heterogeneous nuclear ribonucleoprotein K to VEGF mRNA, which stimulates its translation, was prevented by blockade of AT2, but not AT1. The Akt-mTOR-p70(S6K) signaling pathway, involved in the activation of mRNA translation, was activated in hyperglycemic kidneys and was blocked by the AT2 antagonist. Elongation phase is an important step of mRNA translation that is controlled by elongation factor 1A (eEF1A) and 2 (eEF2). Expression of eEF1A and activity of eEF2 was higher in kidney cortex from hyperglycemic mice and only the AT2 antagonist prevented these changes. To assess selectivity of translational control of VEGF expression, we measured expression of fibronectin (FN) and laminin ß1 (lamß1): acute hyperglycemia increased FN expression at both protein and mRNA levels, indicating transcriptional control, and did not affect the expression of lamß1. To confirm results obtained with PD123319, we induced hyperglycemia in AT2 knockout mice and found that in the absence of AT2, translational control of VEGF expression by hyperglycemia was abolished. Our data show that acute hyperglycemia stimulates Ang II synthesis in murine kidney cortex, this leads to AT2 activation and stimulation of VEGF mRNA translation, via the Akt-mTOR-p70(S6K) signaling pathway. Our data show that exclusive translational control of protein expression in the kidney by acute hyperglycemia is not a general phenomenon, but do not prove that it is restricted to VEGF.

Resveratrol ameliorates high glucose-induced protein synthesis in glomerular epithelial cells.

High glucose-induced protein synthesis in the glomerular epithelial cell (GEC) is partly dependent on reduction in phosphorylation of AMP-activated protein kinase (AMPK). We evaluated the effect of resveratrol, a phytophenol known to stimulate AMPK, on protein synthesis. Resveratrol completely inhibited high glucose stimulation of protein synthesis and synthesis of fibronectin, an important matrix protein, at 3 days. Resveratrol dose-dependently increased AMPK phosphorylation and abolished high glucose-induced reduction in its phosphorylation. We examined the effect of resveratrol on critical steps in mRNA translation, a critical event in protein synthesis. Resveratrol inhibited high glucose-induced changes in association of eIF4E with eIF4G, phosphorylation of eIF4E, eEF2, eEF2 kinase and, p70S6 kinase, indicating that it affects important events in both initiation and elongation phases of mRNA translation. Upstream regulators of AMPK in high glucose-treated GEC were explored. High glucose augmented acetylation of LKB1, the upstream kinase for AMPK, and inhibited its activity. Resveratrol prevented acetylation of LKB1 and restored its activity in high glucose-treated cells; this action did not appear to depend on SIRT1, a class III histone deacetylase. Our data show that resveratrol ameliorates protein synthesis by regulating the LKB1-AMPK axis.

Regulation of mRNA translation in renal physiology and disease.

Translation, a process of generating a peptide from the codons present in messenger RNA, can be a site of independent regulation of protein synthesis; it has not been well studied in the kidney. Translation occurs in three stages (initiation, elongation, and termination), each with its own set of regulatory factors. Mechanisms controlling translation include small inhibitory RNAs such as microRNAs, binding proteins, and signaling reactions. Role of translation in renal injury in diabetes, endoplasmic reticulum stress, acute kidney injury, and, in physiological adaptation to loss of nephrons is reviewed here. Contribution of mRNA translation to physiology and disease is not well understood. Because it is involved in such diverse areas as development and cancer, it should prove a fertile field for investigation in renal science.

PKCdelta regulates the stimulation of vascular endothelial factor mRNA translation by angiotensin II through hnRNP K.

Angiotensin II (Ang II)-induced renal injury is partly mediated by growth factors such as VEGF. We have previously shown that Ang II rapidly increases VEGF protein synthesis in proximal tubular epithelial (MCT) cells by augmenting mRNA translation, which is partly dependent on activation and binding of hnRNP K to 3' untranslated region (UTR) of VEGF mRNA. Regulation of hnRNP K activation by PKCdelta was studied in MCT cells. Transfection with a PKCdelta siRNA inhibited hnRNP K Ser302 phosphorylation and activation, and reduced Ang II stimulation of VEGF synthesis. Inhibition of PKCdelta with röttlerin also prevented binding of hnRNP K to VEGF mRNA and reduced the efficiency of VEGF mRNA translation. In db/db mice at 2 weeks of type 2 diabetes, VEGF expression was increased, which was due not to increase in transcription but to augmented translation of VEGF mRNA. Augmented VEGF expression was associated with increased binding of hnRNP K to VEGF mRNA. c-src and PKCdelta activities and hnRNP K phosphorylation on Ser302 in renal cortex of db/db mice were increased compared to control mice. We conclude: Ang II-induced VEGF mRNA translation is associated with activation of hnRNP K in MCT cells. In the signaling pathway leading to hnRNP K activation induced by Ang II, PKCdelta is downstream of c-src. PKCdelta-mediated phosphorylation of hnRNP K is required for Ang II stimulation of VEGF mRNA translation. In mice with type 2 diabetes, src and PKCdelta activation and hnRNP K phosphorylation correlate with increased VEGF mRNA translation and kidney hypertrophy. 3' UTR events are important in regulation of VEGF expression in models of renal injury.

Regulation of elongation phase of mRNA translation in diabetic nephropathy: amelioration by rapamycin.

High glucose and high insulin, pathogenic factors in type 2 diabetes, induce rapid synthesis of the matrix protein laminin-beta1 in renal proximal tubular epithelial cells by stimulation of initiation phase of mRNA translation. We investigated if elongation phase of translation also contributes to high glucose and high insulin induction of laminin-beta1 synthesis in proximal tubular epithelial cells. High glucose or high insulin rapidly increased activating Thr56 dephosphorylation of eEF2 and inactivating Ser366 phosphorylation of eEF2 kinase, events that facilitate elongation. Studies with inhibitors showed that PI3 kinase-Akt-mTOR-p70S6 kinase pathway controlled changes in phosphorylation of eEF2 and eEF2 kinase induced by high glucose or high insulin. Renal cortical homogenates from db/db mice in early stage of type 2 diabetes showed decrease in eEF2 phosphorylation and increment in eEF2 kinase phosphorylation in association with renal hypertrophy and glomerular and tubular increase in laminin-beta1 content. Rapamycin, an inhibitor of mTOR, abolished diabetes-induced changes in phosphorylation of eEF2, eEF2 kinase, and p70S6 kinase and ameliorated renal hypertrophy and laminin-beta1 protein content, without affecting hyperglycemia. These data show that mTOR is an attractive target for amelioration of diabetes-induced renal injury.

Heterogeneous nuclear ribonucleoprotein K contributes to angiotensin II stimulation of vascular endothelial growth factor mRNA translation.

ANG II rapidly increases VEGF synthesis in proximal tubular epithelial cells through mRNA translation. The role of heterogeneous nuclear ribonucleoprotein K (hnRNP K) in ANG II regulation of VEGF mRNA translation initiation was examined. ANG II activated hnRNP K as judged by binding to poly(C)- and poly(U)-agarose. ANG II increased hnRNP K binding to VEGF mRNA at the same time as it stimulated its translation, suggesting that hnRNP K contributes to VEGF mRNA translation. Inhibition of hnRNP K expression by RNA interference significantly reduced ANG II stimulation of VEGF synthesis. ANG II increased hnRNP K phosphorylation on both tyrosine and serine residues with distinct time courses; only Ser302 phosphorylation paralleled binding to VEGF mRNA. Src inhibition using PP2 or RNA interference inhibited PKCdelta activity and prevented hnRNP K phosphorylation on both tyrosine and serine residues and its binding to VEGF mRNA. Under these conditions, ANG II-induced VEGF synthesis was inhibited. ANG II treatment induced redistribution of both VEGF mRNA and hnRNP K protein from light to heavy polysomal fractions, suggesting increased binding of hnRNP K to VEGF mRNA that is targeted for increased translation. This study shows that hnRNP K augments efficiency of VEGF mRNA translation stimulated by ANG II.

A role for AMP-activated protein kinase in diabetes-induced renal hypertrophy.

We tested the hypothesis that AMP-activated protein kinase (AMPK), an energy sensor, regulates diabetes-induced renal hypertrophy. In kidney glomerular epithelial cells, high glucose (30 mM), but not equimolar mannitol, stimulated de novo protein synthesis and induced hypertrophy in association with increased phosphorylation of eukaryotic initiation factor 4E binding protein 1 and decreased phosphorylation of eukaryotic elongation factor 2, regulatory events in mRNA translation. These high-glucose-induced changes in protein synthesis were phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin (mTOR) dependent and transforming growth factor-beta independent. High glucose reduced AMPK alpha-subunit theronine (Thr) 172 phosphorylation, which required Akt activation. Changes in AMP and ATP content could not fully account for high-glucose-induced reductions in AMPK phosphorylation. Metformin and 5-aminoimidazole-4-carboxamide-1beta-riboside (AICAR) increased AMPK phosphorylation, inhibited high-glucose stimulation of protein synthesis, and prevented high-glucose-induced changes in phosphorylation of 4E binding protein 1 and eukaryotic elongation factor 2. Expression of kinase-inactive AMPK further increased high-glucose-induced protein synthesis. Renal hypertrophy in rats with Type 1 diabetes was associated with reduction in AMPK phosphorylation and increased mTOR activity. In diabetic rats, metformin and AICAR increased renal AMPK phosphorylation, reversed mTOR activation, and inhibited renal hypertrophy, without affecting hyperglycemia. AMPK is a newly identified regulator of renal hypertrophy in diabetes.

mRNA translation: unexplored territory in renal science.

Ambient protein levels are under coordinated control of transcription, mRNA translation, and degradation. Whereas transcription and degradation mechanisms have been studied in depth in renal science, the role of mRNA translation, the process by which peptide synthesis occurs according to the genetic code that is present in the mRNA, has not received much attention. mRNA translation occurs in three phases: Initiation, elongation, and termination. Each phase is controlled by unique eukaryotic factors. In the initiation phase, mRNA and ribosomal subunits are brought together. During the elongation phase, amino acids are added to the nascent peptide chain in accordance with codon sequences in the mRNA. During the termination phase, the fully synthesized peptide is released from the ribosome for posttranslational processing. Signaling pathways figure prominently in regulation of mRNA translation, particularly the phosphatidylinositol 3 kinase-Akt-mammalian target of rapamycin pathway, the AMP-activated protein kinase-tuberous sclerosis complex protein 1/tuberous sclerosis complex protein 2-Rheb pathway, and the extracellular signal-regulated kinase 1/2 type mitogen-activated protein kinase signaling pathway; there is significant cross-talk among these pathways. Regulation by mRNA translation is suggested when changes in mRNA and protein levels do not correlate and in the setting of rapid protein synthesis. Ongoing work suggests an important role for mRNA translation in compensatory renal growth, hypertrophy and extracellular matrix synthesis in diabetic nephropathy, growth factor synthesis by kidney cells, and glomerulonephritis. Considering that mRNA translation plays an important role in cell growth, development, malignancy, apoptosis, and response to stress, its study should provide novel insights in renal physiology and pathology.

Bone morphogenetic protein-7 inhibits constitutive and interleukin-1 beta-induced monocyte chemoattractant protein-1 expression in human mesangial cells: role for JNK/AP-1 pathway.

Bone morphogenetic protein-7 (BMP-7), which belongs to the TGF-beta superfamily, has been shown to reduce macrophage infiltration and tissue injury in animal models of inflammatory renal disease. To explore the mechanism involved in the anti-inflammatory effect, we investigated the effect of BMP-7 on monocyte chemoattractant protein-1 (MCP-1) expression in cultured human mesangial cells. BMP- 7 significantly inhibited constitutive and IL-1 beta-induced MCP-1 protein production and MCP-1 mRNA expression by mesangial cells in a time- and concentration-dependent manner. BMP-7 also inhibited IL-1 beta-induced monocyte chemotactic activity released from the mesangial cells. We examined the role of transcription factors NF-kappa B and AP-1 in BMP-7 inhibition of IL-1 beta-induced MCP-1 expression. IL-1 beta increased NF-kappa B and AP-1 activity and both transcription factors mediated IL-1 beta-induced MCP-1 expression in mesangial cells. BMP-7 inhibited IL-1 beta-induced AP-1 activity in a concentration-dependent manner. In contrast, IL-1 beta-induced NF-kappa B activity and I kappa B alpha degradation were not affected by BMP-7. Furthermore, IL-1 beta-induced phosphorylation of c-Jun N-terminal kinase was inhibited by BMP-7. These data suggest that BMP-7 inhibits constitutive and IL-1 beta-induced MCP-1 expression in human mesangial cells partly by inhibiting c-Jun N-terminal kinase activity and subsequent AP-1 activity, and provide new insight into the therapeutic potential of BMP-7 in the inflammatory renal diseases.

Colchicine decreases apoptotic cell death in chronic cyclosporine nephrotoxicity.

Colchicine has been shown to prevent kidney injury in chronic cyclosporine nephrotoxicity; however, the mechanisms of its action are undetermined. The purpose of this study was to clarify whether colchicine prevents cyclosporine-induced kidney injury by decreasing kidney-cell apoptosis. We also sought to determine whether such an antiapoptotic effect was related to Bcl-2/Bax protein and caspase3 activity. Adult male Sprague-Dawley rats kept on a salt-depleted diet (0.05% sodium) were treated daily for 28 days with cyclosporine (15 mg/kg in 1 mL/kg olive-oil vehicle), colchicine (30 microg/kg in 100% ethanol, diluted with sterile saline solution to a final concentration of 30 microg/mL), or both cyclosporine and colchicine. Kidney function, histomorphologic findings, in situ terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate-biotin nick end-labeling assay, expressions of Bcl-2 and Bax proteins, and caspase-3 enzymatic activity were compared for the different treatment groups. Compared with the vehicle-treated rats, rats given cyclosporine showed a decline in creatinine clearance rate, an increase in serum creatinine concentration, tubulointerstitial fibrosis, and an increase in the number of apoptotic cells (all P <.01). Concomitant administration of colchicine significantly reversed all the above parameters (all P <.05). The decreased expression of Bcl-2 and the ratio of Bcl-2 to Bax protein seen in cyclosporine-treated rat kidneys were significantly increased after colchicine treatment, accompanying a suppression of caspase-3 activity (P <.05). Furthermore, the decreased apoptotic cell death was closely correlated with improved renal tubulointerstitial fibrosis (r = 0.583, P <.05). These findings strongly suggest that a renoprotective effect of colchicine on cyclosporine-induced nephrotoxicity is coassociated with a decrease in apoptotic cells.