Beyond 2D: A scalable and highly sensitive method for a comprehensive 3D analysis of kidney biopsy tissue.

The spatial organization of various cell populations is critical for the major physiological and pathological processes in the kidneys. Most evaluation of these processes typically comes from a conventional 2D tissue cross-section, visualizing a limited amount of cell organization. Therefore, the 2D analysis of kidney biopsy introduces selection bias. The 2D analysis potentially omits key pathological findings outside a 1- to 10-µm thin-sectioned area and lacks information on tissue organization, especially in a particular irregular structure such as crescentic glomeruli. In this study, we introduce an easy-to-use and scalable method for obtaining high-quality images of molecules of interest in a large tissue volume, enabling a comprehensive evaluation of the 3D organization and cellular composition of kidney tissue, especially the glomerular structure. We show that CUBIC and ScaleS clearing protocols could allow a 3D analysis of the kidney tissues in human and animal models of kidney disease. We also demonstrate that the paraffin-embedded human biopsy specimens previously examined via 2D evaluation could be applicable to 3D analysis, showing a potential utilization of this method in kidney biopsy tissue collected in the past. In summary, the 3D analysis of kidney biopsy provides a more comprehensive analysis and a minimized selection bias than 2D tissue analysis. Additionally, this method enables a quantitative evaluation of particular kidney structures and their surrounding tissues, with the potential utilization from basic science investigation to applied diagnostics in nephrology.

Inhibition of Importin- α -Mediated Nuclear Localization of Dendrin Attenuates Podocyte Loss and Glomerulosclerosis.

SIGNIFICANCE STATEMENT: Nuclear translocation of dendrin is observed in injured podocytes, but the mechanism and its consequence are unknown. In nephropathy mouse models, dendrin ablation attenuates proteinuria, podocyte loss, and glomerulosclerosis. The nuclear translocation of dendrin promotes c-Jun N -terminal kinase phosphorylation in podocytes, altering focal adhesion and enhancing cell detachment-induced apoptosis. We identified mediation of dendrin nuclear translocation by nuclear localization signal 1 (NLS1) sequence and adaptor protein importin- α . Inhibition of importin- α prevents nuclear translocation of dendrin, decreases podocyte loss, and attenuates glomerulosclerosis in nephropathy models. Thus, inhibiting importin- α -mediated nuclear translocation of dendrin is a potential strategy to halt podocyte loss and glomerulosclerosis. BACKGROUND: Nuclear translocation of dendrin is observed in the glomeruli in numerous human renal diseases, but the mechanism remains unknown. This study investigated that mechanism and its consequence in podocytes. METHODS: The effect of dendrin deficiency was studied in adriamycin (ADR) nephropathy model and membrane-associated guanylate kinase inverted 2 ( MAGI2 ) podocyte-specific knockout ( MAGI2 podKO) mice. The mechanism and the effect of nuclear translocation of dendrin were studied in podocytes overexpressing full-length dendrin and nuclear localization signal 1-deleted dendrin. Ivermectin was used to inhibit importin- α . RESULTS: Dendrin ablation reduced albuminuria, podocyte loss, and glomerulosclerosis in ADR-induced nephropathy and MAGI2 podKO mice. Dendrin deficiency also prolonged the lifespan of MAGI2 podKO mice. Nuclear dendrin promoted c-Jun N -terminal kinase phosphorylation that subsequently altered focal adhesion, reducing cell attachment and enhancing apoptosis in cultured podocytes. Classical bipartite nuclear localization signal sequence and importin- α mediate nuclear translocation of dendrin. The inhibition of importin- α / ß reduced dendrin nuclear translocation and apoptosis in vitro as well as albuminuria, podocyte loss, and glomerulosclerosis in ADR-induced nephropathy and MAGI2 podKO mice. Importin- α 3 colocalized with nuclear dendrin in the glomeruli of FSGS and IgA nephropathy patients. CONCLUSIONS: Nuclear translocation of dendrin promotes cell detachment-induced apoptosis in podocytes. Therefore, inhibiting importin- α -mediated dendrin nuclear translocation is a potential strategy to prevent podocyte loss and glomerulosclerosis.

IgA-dominant infection-related glomerulonephritis with NAPlr-positive tubulointerstitial nephritis.

Infection-related glomerulonephritis (IRGN) is one of the most common causes of acute kidney injury (AKI). Positive glomerular staining of the nephritis-associated plasmin receptor (NAPlr) has been reported as a useful biomarker of IRGN. Although the infection can provoke acute tubulointerstitial nephritis (AIN), there are few reports of positive staining for NAPlr with AIN. We report a case of methicillin-sensitive Staphylococcus aureus (MSSA) infection-related nephritis complicated with AIN, which showed positive staining for tubulointerstitial NAPlr. The patient developed AKI and nephrotic syndrome during an intraperitoneal MSSA infection. A diagnosis of IRGN complicated by infection-related acute tubulointerstitial nephritis (IRAIN) was made based on glomerular endocapillary proliferation with tubulointerstitial infiltrating cells and tubular atrophy. Tubulointerstitial infiltrating cells were positive for NAPlr staining and plasmin activity. Treatment of the infection by antibiotics and drainage did not improve the AKI, but steroid administration improved that. NAPlr evaluation is a helpful tool for identifying causes of AIN during infection.

Alport syndrome: A case study of chronic type A aortic dissection.

A 39-year-old woman with a history of Alport syndrome was admitted to our hospital for heart failure due to severe aortic regurgitation. Computed tomography revealed a chronic type A aortic dissection that required valve-sparing aortic root replacement. The pathological examination demonstrated that elastic fibers in the tunica media of the aortic wall are torn and severely disorganized. Immunostaining showed fragmented alpha 5 chains, indicating Alport syndrome. These findings imply Alport syndrome may have connective tissue vulnerability, rendering patients susceptible to the development of aortic disease at a young age.

Renal Hyperperfusion Injury After Percutaneous Angioplasty for Renovascular Hypertension as a Sequela of Neuroblastoma.

Hyperperfusion injury is a rare but critical complication associated with revascularization for long-standing severe artery stenosis. Here we report a rare case of a patient with renal hyperperfusion injury after undergoing percutaneous transluminal renal angioplasty for renovascular hypertension as a sequela of neuroblastoma after radiation therapy. (Level of Difficulty: Advanced.).

Impairment of Proteasome Function in Podocytes Leads to CKD.

BACKGROUND: The ubiquitin-proteasome system (UPS) and the autophagy-lysosomal system (APLS) are major intracellular degradation procedures. The importance of the APLS in podocytes is established, but the role of the UPS is not well understood. METHODS: To investigate the role of the UPS in podocytes, mice were generated that had deletion of Rpt3 (Rpt3pdKO), which encodes an essential regulatory subunit required for construction of the 26S proteasome and its deubiquitinating function. RESULTS: Rpt3pdKO mice showed albuminuria and glomerulosclerosis, leading to CKD. Impairment of proteasome function caused accumulation of ubiquitinated proteins and of oxidative modified proteins, and it induced podocyte apoptosis. Although impairment of proteasome function normally induces autophagic activity, the number of autophagosomes was lower in podocytes of Rpt3pdKO mice than in control mice, suggesting the autophagic activity was suppressed in podocytes with impairment of proteasome function. In an in vitro study, antioxidant apocynin and autophagy activator rapamycin suppressed podocyte apoptosis induced by proteasome inhibition. Moreover, rapamycin ameliorated the glomerular injury in the Rpt3pdKO mice. The accumulation of ubiquitinated proteins and of oxidative modified proteins, which were detected in the podocytes of Rpt3pdKO mice, is a characteristic feature of aging. An aging marker was increased in the podocytes of Rpt3pdKO mice, suggesting that impairment of proteasome function promoted signs of aging in podocytes. CONCLUSIONS: Impairment of proteasome function in podocytes led to CKD, and antioxidants and autophagy activators can be therapeutic agents for age-dependent CKD.

Dynamin 1 is important for microtubule organization and stabilization in glomerular podocytes.

Dynamin 1 is a neuronal endocytic protein that participates in vesicle formation by scission of invaginated membranes. Dynamin 1 is also expressed in the kidney; however, its physiological significance to this organ remains unknown. Here, we show that dynamin 1 is crucial for microtubule organization and stabilization in glomerular podocytes. By immunofluorescence and immunoelectron microscopy, dynamin 1 was concentrated at microtubules at primary processes in rat podocytes. By immunofluorescence of differentiated mouse podocytes (MPCs), dynamin 1 was often colocalized with microtubule bundles, which radially arranged toward periphery of expanded podocyte. In dynamin 1-depleted MPCs by RNAi, α-tubulin showed a dispersed linear filament-like localization, and microtubule bundles were rarely observed. Furthermore, dynamin 1 depletion resulted in the formation of discontinuous, short acetylated α-tubulin fragments, and the decrease of microtubule-rich protrusions. Dynamins 1 and 2 double-knockout podocytes showed dispersed acetylated α-tubulin and rare protrusions. In vitro, dynamin 1 polymerized around microtubules and cross-linked them into bundles, and increased their resistance to the disassembly-inducing reagents Ca2+ and podophyllotoxin. In addition, overexpression and depletion of dynamin 1 in MPCs increased and decreased the nocodazole resistance of microtubules, respectively. These results suggest that dynamin 1 supports the microtubule bundle formation and participates in the stabilization of microtubules.

Glomerulosclerosis Induced by Deficiency of Membrane-Associated Guanylate Kinase Inverted 2 in Kidney Podocytes.

Membrane-associated guanylate kinase inverted 2 (MAGI-2) is a component of the slit diaphragm (SD) of glomerular podocytes. Here, we investigated the podocyte-specific function of MAGI-2 using newly generated podocyte-specific MAGI-2-knockout (MAGI-2-KO) mice. Compared with podocytes from wild-type mice, podocytes from MAGI-2-KO mice exhibited SD disruption, morphologic abnormalities of foot processes, and podocyte apoptosis leading to podocyte loss. These pathologic changes manifested as massive albuminuria by 8 weeks of age and glomerulosclerosis and significantly higher plasma creatinine levels at 12 weeks of age; all MAGI-2-KO mice died by 20 weeks of age. Loss of MAGI-2 in podocytes associated with decreased expression and nuclear translocation of dendrin, which is also a component of the SD complex. Dendrin translocates from the SD to the nucleus of injured podocytes, promoting apoptosis. Our coimmunoprecipitation and in vitro reconstitution studies showed that dendrin is phosphorylated by Fyn and dephosphorylated by PTP1B, and that Fyn-induced phosphorylation prevents Nedd4-2-mediated ubiquitination of dendrin. Under physiologic conditions in vivo, phosphorylated dendrin localized at the SDs; in the absence of MAGI-2, dephosphorylated dendrin accumulated in the nucleus. Furthermore, induction of experimental GN in rats led to the downregulation of MAGI-2 expression and the nuclear accumulation of dendrin in podocytes. In summary, MAGI-2 and Fyn protect dendrin from Nedd4-2-mediated ubiquitination and from nuclear translocation, thereby maintaining the physiologic homeostasis of podocytes, and the lack of MAGI-2 in podocytes results in FSGS.

A cell-free method for expressing and reconstituting membrane proteins enables functional characterization of the plant receptor-like protein kinase FERONIA.

There are more than 600 receptor-like kinases (RLKs) in Arabidopsis, but due to challenges associated with the characterization of membrane proteins, only a few have known biological functions. The plant RLK FERONIA is a peptide receptor and has been implicated in plant growth regulation, but little is known about its molecular mechanism of action. To investigate the properties of this enzyme, we used a cell-free wheat germ-based expression system in which mRNA encoding FERONIA was co-expressed with mRNA encoding the membrane scaffold protein variant MSP1D1. With the addition of the lipid cardiolipin, assembly of these proteins into nanodiscs was initiated. FERONIA protein kinase activity in nanodiscs was higher than that of soluble protein and comparable with other heterologously expressed protein kinases. Truncation experiments revealed that the cytoplasmic juxtamembrane domain is necessary for maximal FERONIA activity, whereas the transmembrane domain is inhibitory. An ATP analogue that reacts with lysine residues inhibited catalytic activity and labeled four lysines; mutagenesis demonstrated that two of these, Lys-565 and Lys-663, coordinate ATP in the active site. Mass spectrometric phosphoproteomic measurements further identified phosphorylation sites that were examined using phosphomimetic mutagenesis. The results of these experiments are consistent with a model in which kinase-mediated phosphorylation within the C-terminal region is inhibitory and regulates catalytic activity. These data represent a step further toward understanding the molecular basis for the protein kinase catalytic activity of FERONIA and show promise for future characterization of eukaryotic membrane proteins.

Cell-free translation and purification of Arabidopsis thaliana regulator of G signaling 1 protein.

Arabidopsis thaliana Regulator of G protein Signalling 1 (AtRGS1) is a protein with a predicted N-terminal 7-transmembrane (7TM) domain and a C-terminal cytosolic RGS1 box domain. The RGS1 box domain exerts GTPase activation (GAP) activity on Gα (AtGPA1), a component of heterotrimeric G protein signaling in plants. AtRGS1 may perceive an exogenous agonist to regulate the steady-state levels of the active form of AtGPA1. It is uncertain if the full-length AtRGS1 protein exerts any atypical effects on Gα, nor has it been established exactly how AtRGS1 contributes to perception of an extracellular signal and transmits this response to a G-protein dependent signaling cascade. Further studies on full-length AtRGS1 have been inhibited due to the extreme low abundance of the endogenous AtRGS1 protein in plants and lack of a suitable heterologous system to express AtRGS1. Here, we describe methods to produce full-length AtRGS1 by cell-free synthesis into unilamellar liposomes and nanodiscs. The cell-free synthesized AtRGS1 exhibits GTPase activating activity on Gα and can be purified to a level suitable for biochemical analyses.

Direct injection of cell-free Kir1.1 protein into Xenopus oocytes replicates single-channel currents derived from Kir1.1 mRNA.

The development of integral membrane protein cell-free synthesis permits in-vitro labeling of accessible cysteines for real-time FRET and LRET measurements. The functional integrity of these synthetic ion channel proteins has been verified at the whole oocyte level by direct injection into, and recording from, Xenopus oocytes. However, the microscopic single-channel properties of cell-free translated protein have not been systematically examined. In the present study, we compare patch-clamp currents originating from cell-free protein with currents derived from mRNA injection, using the same (single-Cys) inward rectifier DNA template (C189-Kir1.1b). Results indicate that cell-free Kir protein, incorporated into liposomes and injected into oocytes, is trafficked to the plasma membrane where it inserts in an outside-out orientation and exhibits single-channel characteristics identical to that derived from a corresponding mRNA.

Expression platforms for producing eukaryotic proteins: a comparison of E. coli cell-based and wheat germ cell-free synthesis, affinity and solubility tags, and cloning strategies.

Vectors designed for protein production in Escherichia coli and by wheat germ cell-free translation were tested using 21 well-characterized eukaryotic proteins chosen to serve as controls within the context of a structural genomics pipeline. The controls were carried through cloning, small-scale expression trials, large-scale growth or synthesis, and purification. Successfully purified proteins were also subjected to either crystallization trials or (1)H-(15)N HSQC NMR analyses. Experiments evaluated: (1) the relative efficacy of restriction/ligation and recombinational cloning systems; (2) the value of maltose-binding protein (MBP) as a solubility enhancement tag; (3) the consequences of in vivo proteolysis of the MBP fusion as an alternative to post-purification proteolysis; (4) the effect of the level of LacI repressor on the yields of protein obtained from E. coli using autoinduction; (5) the consequences of removing the His tag from proteins produced by the cell-free system; and (6) the comparative performance of E. coli cells or wheat germ cell-free translation. Optimal promoter/repressor and fusion tag configurations for each expression system are discussed.

Automated cell-free protein production methods for structural studies.

In contrast to cell-based protein expression, cell-free production is highly consistent, scalable, and amenable to automation. Robots can handle many samples and perform repetitive procedures that are otherwise prone to human error. Here is described commercially available robotics for a wheat germ cell-free system with emphasis on practical applications for structural and functional studies. In addition, described is a cell-free method for preparing protein complexes.

Cell-free translation of biofuel enzymes.

In nature, bacteria and fungi are able to utilize recalcitrant plant materials by secreting a diverse set of enzymes. While genomic sequencing efforts offer exhaustive lists of genes annotated as potential polysaccharide-degrading enzymes, biochemical and functional characterizations of the encoded proteins are still needed to realize the full potential of this natural genomic diversity. This chapter outlines an application of wheat germ cell-free translation to the study of biofuel enzymes using genes from Clostridium thermocellum, a model cellulolytic organism. Since wheat germ extract lacks enzymatic activities that can hydrolyze insoluble polysaccharide substrates and is likewise devoid of enzymes that consume the soluble sugar products, the cell-free translation reactions provide a clean background for production and study of the reactions of biofuel enzymes. Examples of assays performed with individual enzymes or with small sets of enzymes obtained directly from cell-free translation are provided.

Cell-free protein synthesis for functional and structural studies.

Recent advances in cell-free protein expression systems have made them reliable and practical for functional and structural studies of a wide variety of proteins. In particular, wheat germ cell-free translation can consistently produce target proteins in microgram quantities from relatively inexpensive, small-scale reactions. Here we describe our small-scale protein expression method for rapidly producing proteins for functional assay and techniques for determining if the target is suitable for scale-up to amounts potentially needed for structure determination. The cell-free system is versatile and can be easily customized with the inclusion of additives. We describe simple modifications used for producing membrane proteins.

Structural and functional characterization of CalS11, a TDP-rhamnose 3'-O-methyltransferase involved in calicheamicin biosynthesis.

Sugar methyltransferases (MTs) are an important class of tailoring enzymes that catalyze the transfer of a methyl group from S-adenosyl-l-methionine to sugar-based N-, C- and O-nucleophiles. While sugar N- and C-MTs involved in natural product biosynthesis have been found to act on sugar nucleotide substrates prior to a subsequent glycosyltransferase reaction, corresponding sugar O-methylation reactions studied thus far occur after the glycosyltransfer reaction. Herein we report the first in vitro characterization using (1)H-(13)C-gHSQC with isotopically labeled substrates and the X-ray structure determination at 1.55 Å resolution of the TDP-3'-O-rhamnose-methyltransferase CalS11 from Micromonospora echinospora. This study highlights a unique NMR-based methyltransferase assay, implicates CalS11 to be a metal- and general acid/base-dependent O-methyltransferase, and as a first crystal structure for a TDP-hexose-O-methyltransferase, presents a new template for mechanistic studies and/or engineering.

Mutations in FLS2 Ser-938 dissect signaling activation in FLS2-mediated Arabidopsis immunity.

Flagellin-sensing 2 (FLS2) is a leucine-rich repeat/transmembrane domain/protein kinase (LRR-RLK) that is the plant receptor for bacterial flagellin or the flagellin-derived flg22 peptide. Previous work has shown that after flg22 binding, FLS2 releases BIK1 kinase and homologs and associates with BAK1 kinase, and that FLS2 kinase activity is critical for FLS2 function. However, the detailed mechanisms for activation of FLS2 signaling remain unclear. The present study initially identified multiple FLS2 in vitro phosphorylation sites and found that Serine-938 is important for FLS2 function in vivo. FLS2-mediated immune responses are abolished in transgenic plants expressing FLS2(S938A), while the acidic phosphomimic mutants FLS2(S938D) and FLS2(S938E) conferred responses similar to wild-type FLS2. FLS2-BAK1 association and FLS2-BIK1 disassociation after flg22 exposure still occur with FLS2(S938A), demonstrating that flg22-induced BIK1 release and BAK1 binding are not sufficient for FLS2 activity, and that Ser-938 controls other aspects of FLS2 activity. Purified BIK1 still phosphorylated purified FLS2(S938A) and FLS2(S938D) mutant kinase domains in vitro. Phosphorylation of BIK1 and homologs after flg22 exposure was disrupted in transgenic Arabidopsis thaliana plants expressing FLS2(S938A) or FLS2(D997A) (a kinase catalytic site mutant), but was normally induced in FLS2(S938D) plants. BIK1 association with FLS2 required a kinase-active FLS2, but FLS2-BAK1 association did not. Hence FLS2-BIK1 dissociation and FLS2-BAK1 association are not sufficient for FLS2-mediated defense activation, but the proposed FLS2 phosphorylation site Ser-938 and FLS2 kinase activity are needed both for overall defense activation and for appropriate flg22-stimulated phosphorylation of BIK1 and homologs.

Function of Shaker potassium channels produced by cell-free translation upon injection into Xenopus oocytes.

Voltage-gated ion channels are a class of membrane proteins that temporally orchestrate the ion flux critical for chemical and electrical signaling in excitable cells. Current methods to investigate the function of these channels rely on heterologous expression in living systems or reconstitution into artificial membranes; however these approaches have inherent drawbacks which limit potential biophysical applications. Here, we describe a new integrated approach combining cell-free translation of membrane proteins and in vivo expression using Xenopus laevis oocytes. In this method, proteoliposomes containing Shaker potassium channels are synthesized in vitro and injected into the oocytes, yielding functional preparations as shown by electrophysiological and fluorescence measurements within few hours. This strategy for studying eukaryotic ion channels is contrasted with existing, laborious procedures that require membrane protein extraction and reconstitution into synthetic lipid systems.

Cell-free production of integral membrane aspartic acid proteases reveals zinc-dependent methyltransferase activity of the Pseudomonas aeruginosa prepilin peptidase PilD.

Integral membrane aspartic acid proteases are receiving growing recognition for their fundamental roles in cellular physiology of eukaryotes and prokaryotes, and may be medically important pharmaceutical targets. The Gram-negative Pseudomonas aeruginosa PilD and the archaeal Methanococcus voltae FlaK were synthesized in the presence of unilamellar liposomes in a cell-free translation system. Cosynthesis of PilD with its full-length substrate, PilA, or of FlaK with its full-length substrate, FlaB2, led to complete cleavage of the substrate signal peptides. Scaled-up synthesis of PilD, followed by solubilization in dodecyl-ß-d-maltoside and chromatography, led to a pure enzyme that retained both of its known biochemical activities: cleavage of the PilA signal peptide and S-adenosyl methionine-dependent methylation of the mature pilin. X-ray fluorescence scans show for the first time that PilD is a zinc-binding protein. Zinc is required for the N-terminal methylation of the mature pilin, but not for signal peptide cleavage. Taken together, our work identifies the P. aeruginosa prepilin peptidase PilD as a zinc-dependent N-methyltransferase and provides a new platform for large-scale synthesis of PilD and other integral membrane proteases important for basic microbial physiology and virulence.

Application of the wheat-germ cell-free translation system to produce high temperature requirement A3 (HtrA3) proteases.

Mammalian high temperature requirement A3 (HtrA3) is a serine protease of the HtrA family. It is an important factor for placental development and a tumor suppressor. The biochemical properties of HtrA3 are uncharacterized. One critical step in biochemical characterization is overexpressing and purifying the full-length recombinant protein. However, utility of cell-based expression systems is limited for a protease because of autocleavage. The wheat-germ cell-free translation system is highly efficient at producing "difficult" eukaryotic multidomain proteins and is easily modifiable for protein synthesis at different temperatures. In this study, we evaluated the potential of the wheat-germ cell-free translation system for producing human HtrA3. HtrA3 underwent autocleavage when synthesized at 17 °C. When the synthesis temperature was lowered to 4 °C, full-length HtrA3 was successfully produced and proteolytically active. Catalytic site serine substitution with alanine (S305A) stabilized HtrA3 while abolishing its protease activity. This mutant was readily synthesized and stable at 17 °C. When used with glutathione S-transferase (GST) pull-down assay, S305A HtrA3 was a valuable bait in searching for endogenous HtrA3 binding proteins. Thus, we demonstrated the unique utility of the wheat-germ cell-free translation system for producing and characterizing human HtrA3. These strategies will be likely applicable to a wide range of proteases.