Secondary Foot Research Articles

#5919 PATIENT-SPECIFIC HUMAN INDUCED PLURIPOTENT STEM CELL (HIPSC)-DERIVED PODOCYTES

Abstract Background and Aims Podocytes typically represent a distinct morphology with long, interdigitating primary and secondary foot processes wrapping around glomerular capillaries forming the filtration unit of the kidney. In glomerular diseases like genetic focal segmental glomerulosclerosis (FSGS), podocyte alterations are often related to dysfunction of the glomerular filtration barrier and the development of proteinuria. Genetic mutations in podocyte relevant genes, important for foot process or actin cytoskeleton dynamics, are known to induce severe podocyte injury. Analysis of podocyte alterations ex vivo is limited because of their inability to proliferate due to their terminal differentiated state and limited access to primary podocytes. Instead, immortalized podocytes regained proliferation capacity by insertion of a thermosensitive SV40 large T antigen and are useful for many research questions. However, these podocytes usually do not express all podocyte marker and foot process development is often restricted. Since we aim to characterize personalized podocytes from patients suffering from genetic FSGS ex vivo, we generated podocytes from dermal fibroblasts via reprogramming into hiPSCs and subsequent differentiation into hiPSC-derived podocytes keeping the patients’ genetic background. Method Dermal fibroblasts were outgrown from a skin punch biopsy from a patient or a healthy donor and subsequently reprogrammed into hiPSCs by electroporation of the common OKSM plasmids and finally differentiated into podocytes. Cells were characterized phenotypically regarding morphology and marker expression using RNA bulk sequencing, western blot, qPCR and immunofluorescence staining. Potential functional alterations of diseased podocytes regarding actin cytoskeleton were assessed by actin polymerization assay. Results Patient-specific podocytes could be generated ex vivo by reprogramming dermal fibroblasts into hiPSCs and subsequently differentiating them into mature podocytes representing the patients’ background. During this process, cell morphology changed from spindle-like fibroblasts to small and round hiPSCs growing in colonies and representing a high nuclei-to body ratio as well as high proliferation rate. During differentiation, hiPSCs were induced into intermediate mesoderm, resulting in increased cell size and decreased proliferation rate terminating in 300 nm large star-shaped podocytes developing a distinct network of long primary and secondary foot processes. Patient-derived hiPSC-podocytes with a mutation in the INF2 gene lacked filamentous actin and only develop a limited number of only short foot processes compared to hiPSC-podocytes derived from a healthy control donor. Moreover, expression of actin cytoskeleton associated genes like SYNPO, ACTN4 and CD2AP was decreased while other actin isoforms, cortactin and the GTPase RhoA were increased in diseased patient-specific podocytes. Actin polymerization assay showed that polymerization process itself was not altered in healthy and diseased hiPSC-podocytes. Conclusion Analysis of patient-specific podocytes is possible ex vivo via differentiation of hiPSCs, generated from patients’ dermal fibroblasts by episomal reprogramming, into hiPSC-derived podocytes, with the potential to study alterations of disease-specific mutations regarding phenotypical and functional behavior in a personalized manner. The use of hiPSCs bypasses the limitation of restricted podocyte cell number and has the advantage of maintaining the patients’ genetic background at podocyte cell level. This enables future large-scale experiments regarding intercell-cell communication and interaction in glomerular three-dimensional co-cultures or via treatment with therapeutic substances or stress factors.

Characterizing Intraindividual Podocyte Morphology In Vitro with Different Innovative Microscopic and Spectroscopic Techniques.

Podocytes are critical components of the glomerular filtration barrier, sitting on the outside of the glomerular basement membrane. Primary and secondary foot processes are characteristic for podocytes, but cell processes that develop in culture were not studied much in the past. Moreover, protocols for diverse visualization methods mostly can only be used for one technique, due to differences in fixation, drying and handling. However, we detected by single-cell RNA sequencing (scRNAseq) analysis that cells reveal high variability in genes involved in cell type-specific morphology, even within one cell culture dish, highlighting the need for a compatible protocol that allows measuring the same cell with different methods. Here, we developed a new serial and correlative approach by using a combination of a wide variety of microscopic and spectroscopic techniques in the same cell for a better understanding of podocyte morphology. In detail, the protocol allowed for the sequential analysis of identical cells with light microscopy (LM), Raman spectroscopy, scanning electron microscopy (SEM) and atomic force microscopy (AFM). Skipping the fixation and drying process, the protocol was also compatible with scanning ion-conductance microscopy (SICM), allowing the determination of podocyte surface topography of nanometer-range in living cells. With the help of nanoGPS Oxyo®, tracking concordant regions of interest of untreated podocytes and podocytes stressed with TGF-β were analyzed with LM, SEM, Raman spectroscopy, AFM and SICM, and revealed significant morphological alterations, including retraction of podocyte process, changes in cell surface morphology and loss of cell-cell contacts, as well as variations in lipid and protein content in TGF-β treated cells. The combination of these consecutive techniques on the same cells provides a comprehensive understanding of podocyte morphology. Additionally, the results can also be used to train automated intelligence networks to predict various outcomes related to podocyte injury in the future.

Protective effects of (4-(1,2,4-oxadiazol-5-yl)phenyl)-2-aminoacetamide derivatives to adjuvant-induced arthritis rats by regulating the NF-κB signaling pathway.

A series of (4-(1,2,4-oxadiazol-5-yl) phenyl)-2-aminoacetamide derivatives showed good anti-neuroinflammation in our previous study. Some studies have proven that the anti-inflammatory compounds effective for some specific diseases could also be used to treat other inflammatory diseases. In this study, the effects of these compounds on arthritis were further analyzed. First, in-vitro anti-inflammatory activity assessment indicated that these compounds have good anti-inflammatory activity. Among them, compound f15 showed the most prominent performance, it could significantly inhibit the production of relevant inflammatory factors in lipopolysaccharide (LPS)-induced RAW264.7 cells, with IC50 values of 1.55 ± 0.33, 3.83 ± 0.19, and 7.03 ± 0.24μM against NO, IL-1β, and TNF-α production, respectively. Preliminary mechanism studies indicated that f15 blocked the excitation of nuclear factor κB (NF-кB) signaling pathway in a concentration-dependent manner. Furthermore, in-vivo experiment showed that f15 reduced secondary foot swelling and arthritic index in adjuvant-induced arthritis (AIA) rats and inhibited the production of TNF-α and IL-1β in serum. Histopathological analysis revealed that f15 alleviated inflammatory cell infiltration and synovial hyperplasia in rats with AIA. Thus, compound f15 could be considered to have the potential to be developed as a treatment for arthritis.

Celastrol Exerts Cardioprotective Effect in Rheumatoid Arthritis by Inhibiting TLR2/HMGB1 Signaling Pathway-Mediated Autophagy

Objective: Rheumatoid arthritis (RA) is a kind of chronic inflammatory disease characterized by the release of inflammatory cytokines and cardiomyocyte apoptosis, which lead to increased riskfor heart diseases. This study aims to explore the possible effect and mechanism of Celastrol on RA induced cardiac impairments in rats. Methods: Collagen induced RA wistar rat models (CIA) were established for the measurement on secondary foot swelling degree, polyarthritis index score, spleen and thymus index. Pathological morphology was observed using H&E staining. Heart fibrosis was measured after Sirius red staining, while cell apoptosis was determined by TUNEL staining. For in vitro experiments, rat cardiomyocytes were isolated to determine the inflammatory cytokine secretion and cell apoptosis using ELISA and flow cytometry, respectively. Protein expressions of related index and autophagy were detected by Western blot and immunofluorescence. Results: CIA rat model was successfully established and characterized by severe secondary foot swelling degree, and increased polyarthritis index score and spleen and thymus index. Synovial hyperplasia, disordered cardiomyocytes, cell infiltration and fibrosis were also observed in CIA rat model. Compared with CIA model, Celastrol treatment could suppress the release of inflammatory cytokines, including TNF-α, IL-6, IL-1β, as well as inhibiting the expressions of Bax, cleaved caspase3, collagen I, collagen III and α-SMA. In addition to that, Celastrol treatment can attenuate cell apoptosis and fibrosis of cardiomyocytes and elevate Bcl-2 expression. RA induced cell autophagy can be suppressed by Celastrol through inhibiting the activation of TLR2/HMGB1 signal pathway. Conclusion: Celastrol can regulate TLR2/HMGB1 signal pathway to suppress autophagy and therefore exert cardioprotective effect in RA.

Hh pathway expression in the blood, synovial cells and chondrocytes of different rheumatoid arthritis models

Purpose: To investigate the effect of the hedgehog (Hh) pathway inhibitor, cyclopamine, and activator purmorphamine on articular cartilage cell proliferation.
 Methods: Rats were subjected to AA and CIA models. Secondary paw swelling was measured at 12, 15, 18, 21, 24, 27, and 30 days. The rats were sacrificed on day 30. Tissues from the cartilage and knee joints were collected. Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay while cell apoptosis was determined by annexin V-fluorescein isothiocyanate/propidium iodide assay. Protein expression levels of Shh, Ptch1 and Gli1 were determined by Western blotting.
 Results: Compared with the control group, arthritis index and secondary foot swelling of the adjuvant arthritis (AA) and collagen-induced arthritis (CIA) groups deteriorated significantly (p < 0.05). MTT data revealed that cyclopamine promoted articular cartilage cell proliferation of the AA and CIA groups. The cell proliferation rates of AA and CIA groups were significantly higher than that of control group (p < 0.05). Flow cytometry showed that the cell apoptosis rates of AA and CIA groups were significantly lower than that of control group (p < 0.05). Compared with control, cyclopamine decreased the protein expression levels of sonic Hh, patched homologue 1 and glioma-associated oncogene homologue, but the effect of purmorphamine was the reverse.
 Conclusion: Hh pathway inhibitor (cyclopamine) and activator (purmorphamine) affect the expression of Hh pathway. Disruption of the Hh pathway may be of potential therapeutic significance in protecting articular cartilage from rheumatoid arthritis.

Finite Element Analysis of Generalized Ligament Laxity on the Deterioration of Hallux Valgus Deformity (Bunion).

Hallux valgus is a common foot problem affecting nearly one in every four adults. Generalized ligament laxity was proposed as the intrinsic cause or risk factor toward the development of the deformity which was difficult to be investigated by cohort clinical trials. Herein, we aimed to evaluate the isolated influence of generalized ligament laxity on the deterioration using computer simulation (finite element analysis). We reconstructed a computational foot model from a mild hallux valgus participant and conducted a gait analysis to drive the simulation of walking. Through parametric analysis, the stiffness of the ligaments was impoverished at different degrees to resemble different levels of generalized ligament laxity. Our simulation study reported that generalized ligament laxity deteriorated hallux valgus by impairing the load-bearing capacity of the first metatarsal, inducing higher deforming force, moment and malalignment at the first metatarsophalangeal joint. Besides, the deforming moment formed a deteriorating vicious cycle between hallux valgus and forefoot abduction and may result in secondary foot problems, such as flatfoot. However, the metatarsocuneiform joint did not show a worsening trend possibly due to the overriding forefoot abduction. Controlling the deforming load shall be prioritized over the correction of angles to mitigate deterioration or recurrence after surgery.

Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions.

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney disease and eventual end-stage renal failure are often caused by the damage to the glomerular podocytes, which are the highly specialized epithelial cells that function together with endothelial cells and the glomerular basement membrane to form the kidney's filtration barrier. Advances in renal medicine have been hindered by the limited availability of primary tissues and the lack of robust methods for the derivation of functional human kidney cells, such as podocytes. The ability to derive podocytes from renewable sources, such as stem cells, could help advance current understanding of the mechanisms of human kidney development and disease, as well as provide new tools for therapeutic discovery. The goal of this protocol was to develop a method to derive mature, post-mitotic podocytes from human induced pluripotent stem (hiPS) cells with high efficiency and specificity, and under chemically defined conditions. The hiPS cell-derived podocytes produced by this method express lineage-specific markers (including nephrin, podocin, and Wilm's Tumor 1) and exhibit the specialized morphological characteristics (including primary and secondary foot processes) associated with mature and functional podocytes. Intriguingly, these specialized features are notably absent in the immortalized podocyte cell line widely used in the field, which suggests that the protocol described herein produces human kidney podocytes that have a developmentally more mature phenotype than the existing podocyte cell lines typically used to study human kidney biology.

Analysis of the Possibilities of Using a Driver’s Brain Activity to Pneumatically Actuate a Secondary Foot Brake Pedal

The study deals with the use of the driver’s brain activity for wireless remote control of the pneumatic actuator exerting pressure on the secondary foot brake pedal. The conducted experimental tests confirm that bioelectrical signals (BES) induced by muscle tension within the head can be used for wireless remote control of a pneumatic actuator to exert a pressure force on a foot brake pedal for disabled drivers during car emergency braking. It has been shown that the BES artefacts generated by muscular tension inside the head (e.g., movement of the face and eyelids, clenching of jaws, and pressing the tongue on the palate) are the easiest to control of the pneumatic systems. The proposed car braking assistance system controlled by the driver’s brain activity can improve the driving safety of disabled people, e.g., by reducing the reaction time of pneumatically assisted emergency braking.

Improvement in activities and participation in an adolescent following secondary foot amputation

Abstract Background When severe limb trauma occurs in a child, all efforts are made to preserve the limb as far as possible. In rare cases, however, secondary amputation may be necessary to improve functional outcome and participation. Case report In this paper, we report on a young adolescent male with a severe loss of function following foot trauma, who underwent secondary trans-tibial amputation 17 months post injury. This paper describes how a structured multidisciplinary approach around the child and his family led to a successful outcome. Clinical rehabilitation impact It is hoped that this case report may help other health professionals with decision-making in cases of secondary amputations, and to implement appropriate follow-up for such patients.

LIM and SH3 protein 1 (LASP-1): A novel link between the slit membrane and actin cytoskeleton dynamics in podocytes.

The foot processes of podocytes exhibit a dynamic actin cytoskeleton, which maintains their complex cell structure and antagonizes the elastic forces of the glomerular capillary. Interdigitating secondary foot processes form a highly selective filter for proteins in the kidney, the slit membrane. Knockdown of slit membrane components such as Nephrin or Neph1 and cytoskeletal adaptor proteins such as CD2AP in mice leads to breakdown of the filtration barrier with foot process effacement, proteinuria, and early death of the mice. Less is known about the crosstalk between the slit membrane-associated proteins and cytoskeletal components inside the podocyte foot processes. Our study shows that LASP-1, an actin-binding protein, is highly expressed in podocytes. Electron microscopy studies demonstrate that LASP-1 is found at the slit membrane suggesting a role in anchoring slit membrane components to the actin cytoskeleton. Live cell imaging experiments with transfected podocytes reveal that LASP-1iseither part of a highly dynamic granular complex or a static, actin cytoskeleton-bound protein. We identify CD2AP as a novel LASP-1 binding partner that regulates its association with the actin cytoskeleton. Activation of the renin-angiotensin-aldosterone system, which is crucial for podocyte function, leads to phosphorylation and altered localization of LASP-1. In vivo studies using the Drosophila nephrocyte model indicate that Lasp is necessary for the slit membrane integrity and functional filtration.

Biochemical and histological study on the effect of levetiracetam on the liver and kidney of pregnant albino rats

Levetiracetam is a broad-spectrum antiseizure agent and one of the most commonly prescribed drugs for epilepsy. The aim of this work was to assess the effect of levetiracetam at its therapeutic range on the liver and kidney of pregnant albino rats. Forty pregnant rats were divided equally into two groups (I-II), Rats in the group I were gavaged 1.5 mL/day distilled water in two divided doses throughout pregnancy. Rats in the group II were gavaged 1.5 mL/day distilled water (containing 36 mg levetiracetam) in two divided doses throughout pregnancy. At the end of the experiment, blood samples were taken and the sera were separated and used for biochemical analysis. The kidneys and livers of both groups were excised and used for light and electron microscopic examination. Treatment with levetiracetam induced undesirable histopathological changes in the liver and kidney of pregnant albino rats. These changes were in the form of distortion of the hepatic architecture, dilatation of the central and the portal veins, widening of the Bowman's spaces, thickening and disruption of the glomerular basement membrane, fusion and effacement of secondary foot processes, cytoplasmic vacuolation, and swollen mitochondria with loss of their cristae. Such changes were confirmed by alteration of certain biochemical parameters related to the liver and kidney functions. Levetiracetam induced deleterious effects on the liver and kidney of pregnant albino rats. Further investigations are recommended to clarify the mechanism of levetiracetam toxicity.

SAT-115 Generation and characterisation of induced pluripotent stem cells derived from patients with Fabry disease

Fabry disease (FD) is an X-linked inherited disorder resulting in accumulation of globotriaosylceramide (Gb3) that ultimately progresses to end-stage renal disease. Although the genetic basis of FD is well established, the pathophysiological mechanisms by which Gb3 storage leads to the FD phenotype remains unclear. It is well defined that the Gb3 accumulation in FD causes podocyte damage leading to fibrosis. To date, modelling of renal diseases and drug screening has utilised immortalised kidney cell lines which retain only limited characteristics of their somatic cell counterparts. Alternatively, primary podocytes are difficult to establish and maintain in culture. We therefore generated induced pluripotent stem cells (iPSC) by reprogramming human somatic cells from FD patients, with varying known FD mutations, to generate iPSCs followed by directed differentiation to podocytes that can be maintained long-term. Skin fibroblasts obtained from punch biopsies of patients with mapped FD mutations (n=3) compared to non-FD control patients (n=3) were obtained under informed consent. A NM-RNA reprogramming protocol that delivers transcription factors OCT3/4, TRA-1-60 and SSEA4 in lipofectamines through RNA transfection, was used to generate iPSCs. Pluripotency was confirmed by alkaline phosphatase staining and immunofluorescence staining performed using Oct4 and CSTEM 27, 28, 31. FD and control iPSCs were differentiated to podocytes following the addition of retinoic acid, bone morphogenic protein 7 and activin A. Day 10 iPSC-podocytes were immunostained for the expression of podocyte-specific markers – podocin and synaptopodin. Scanning electron microscopy (SEM) was performed on FD patient-derived iPSCs and control lines to observe podocyte morphological following differentiation and quantification of Gb3 performed using mass spectrometry (MS). Skin biopsy-derived dermal fibroblasts from FD and non-FD control patients were grown as monolayers and reprogrammed into iPSCs over 14 days, with pluripotency evident by alkaline phosphatase staining with normal karyotype confirmed. Colonies showed positive expression of pluripotency markers - Oct4 and CSTEM 27, 28 and 31. Directed differentiation of iPSCs resulted in the generation of podocytes over a period of 10 days that had an acquired typical podocyte morphology, consisting of either a single or multi-nucleated cell bodies and prominent foot processes. Day 10 iPSC-podocytes showed positive expression of podocyte-specific markers - podocin and synaptopodin. SEM imaging of differentiated iPSC-podocytes revealed primary and secondary foot processes, characteristic of kidney podocytes with no major morphological differences apparent between FD and control lines. Gb3 quantification using MS showed a varied increase in FD iPSC podocytes reflective of varied mutations associated with a range of pathologies and severity of disease, compared to non-FD cells. This study has generated iPSC from patients with FD that were differentiated to podocytes confirmed by morphological characteristics and podocyte marker localization. Understanding how mutations cause disease in FD using iPSC-derived kidney cells is a fundamental step for future studies to correct the genetic defects in these cells and as a valuable tool for use in disease modeling and toxicology screening.

Impact of renal ischemia/reperfusion injury on the rat Kupffer cell as a remote cell: A biochemical, histological, immunohistochemical, and electron microscopic study

Almost all transplanted solid organs are exposed to some degree of ischemia-reperfusion (IR) damage. It is interesting to know that this IR damage affects various remote tissues including the liver and resulted in serious adverse effects. Liver injury triggers different responses of liver tissue especially Kupffer cells (KCs). The goal of this current study is to assess the biochemical and morphological changes of hepatic KCs after the induction of renal ischemia-reperfusion (RIR) and point out their role in remote liver injury after RIR.Sixteen male Sprague-Dawley rats were randomly divided into two equal groups: Group I; sham group. Group II; renal ischemia reperfusion (IR) group in which rats were exposed to renal ischemia for 45 min followed by renal reperfusion for 48 h. Three rats from each group were subjected to charcoal injection to evaluate KCs activity. Specimens of rat liver from each group were obtained and processed for biochemical, light microscopic and ultramicroscopic examination. The current results showed elevated serum levels of AST and ALT. The liver HGF-α protein expression increased in IR group compared to the sham group. In IR group, numerous charcoal labeled KCs were observed mainly localized around the central vein. Scanning electron micrographs showed complex primary and secondary foot process of the KCs. Ultrastructural study showed KCs with multiple cytoplasmic vacuoles, lysosomes and mitochondria, rough endoplasmic reticulum and ribosomes. Immuno-histochemical study showed more tumor necrosis factor-α (TNF-α) expression in KCs than the sham group. These results collectively demonstrated that renal IR produced biochemical and morphological changes in the liver KCs and theses cells might have a role in the remote liver injury after renal IR. This might be one of the mechanisms through which RIR affects the liver.

MiR-125 regulates PI3K/Akt/mTOR signaling pathway in rheumatoid arthritis rats via PARP2.

The present study aimed to explore miR-125 effects on rheumatoid arthritis (RA) development to provide a potential target for RA. Briefly, rat RA model was established (Model group) by injection of Freund’s Complete Adjuvant into the left hind toe. Normal rats injected with saline in the same location were set as Normal group. All rats’ secondary foot swelling degree, polyarthritis index score, spleen and thymus index were measured. Synovial tissues were subjected to Hematoxylin–Eosin (HE) staining and immunohistochemistry. Synovial cells of each group were isolated and named as Normal-C group and Model-C group, respectively. Synovial cells of Model-C group further underwent cotransfection with miR-125 mimics and PARP2-siRNA (mimics+siPARP2 group) or with miR-125 negative control (NC) and PARP2-siRNA NC (NC group). Quantitative reverse transcriptase PCR (qRT-PCR), Western blot, luciferase reporter assay, ELISA, and MTT assay were performed. As a result, compared with Normal group, rats of Model group showed significantly higher secondary foot swelling degree, polyarthritis index score, spleen and thymus index (P<0.01). Down-regulated miR-125 and up-regulated PARP2 was found in synovial tissues of Model group when compared with Normal group (P<0.01). Synovial tissues of Model-C group exhibited severe hyperplasia and inflammatory cell infiltration. Luciferase reporter assay indicated that PARP2 was directly inhibited by miR-125. Compared with NC group, cells of mimics+siPARP2 group had significantly lower IL-1β, MMP-1 and TIMP-1 levels, absorbance value, and p-PI3K, p-Akt and p-mTOR relative expression (P<0.01 or P<0.05). Thus, miR-125 might attenuate RA development by regulating PI3K/Akt/mTOR signaling pathway via directly inhibiting PARP2 expression.

MICROSCOPICAL STUDIES OF THE EFFECT OF THE ORGANOPHOSPHOROUS INSECTICIDE MONOCROTOPHOS ON THE KIDNEYS OF MALE ALBINO MICE

The present study aimed to throw more light on the effect of the insecticide monocrotophos on the kidneys of mice treated orally with the insecticide. The applied doses were 1/20, 1/6 and 1/3 LD50 (0.38, 1.25 and 2.5 mg/kg body weight), respectively. Each dose was given for seven successive days. The observed histopathological and ultrastructural alterations were found to be dose-dependent. The low dose (1/20 LD50) caused shrinkage of some glomeruli; some were congested and the cellularity in some others were increased. Both of the proximal and distal convoluted tubules cells showed many cytoplasmic vacuoles and pyknotic nuclei and disappearance of microvilli of the proximal convoluted tubule cells. The degenerative alterations became more severe in case of the median (1/6 LD50) and the high (1/3 LD50) doses of monocrotophos. In such cases, the glomeruli revealed cellular abnormalities, necrosis and some of them were deeply clefted. Dissociated and degenerated renal tubule cells with pyknotic nuclei, and cell debris in the tubules lumina were observed in the kidney cortex. In case of the cumulative treatment, monocrotophos in a dose of 0.19 mg/kg given for twenty successive day, caused similar degenerative changes to the afore-mentioned alterations. These histopathological alterations showed partial recovery when inspected on the seventh day after the last dose of treatment, and the recovery became more pronounced on the fourteenth days after the last treatment. This may indicate withdrawal of the insecticide from the experimental animal after stopping treatment. The ultrastructural alterations showed increased mesangial matrix and atrophied podocytes with fusion of their secondary foot processes. The cells of the convoluted renal tubules displayed degenerated mitochondria, short profiles of RER, pyknotic and chromatolytic nuclei and presence of cytosegrosomes (sequestration of parts of their cytoplasm).

Mature induced-pluripotent-stem-cell-derived human podocytes reconstitute kidney glomerular-capillary-wall function on a chip

An in vitro model of the human kidney glomerulus — the major site of blood filtration — could facilitate drug discovery and illuminate kidney-disease mechanisms. Microfluidic organ-on-a-chip technology has been used to model the human proximal tubule, yet a kidney-glomerulus-on-a-chip has not been possible because of the lack of functional human podocytes — the cells that regulate selective permeability in the glomerulus. Here, we demonstrate an efficient (> 90%) and chemically defined method for directing the differentiation of human induced pluripotent stem (hiPS) cells into podocytes that express markers of the mature phenotype (nephrin+, WT1+, podocin+, Pax2−) and that exhibit primary and secondary foot processes. We also show that the hiPS-cell-derived podocytes produce glomerular basement-membrane collagen and recapitulate the natural tissue/tissue interface of the glomerulus, as well as the differential clearance of albumin and inulin, when co-cultured with human glomerular endothelial cells in an organ-on-a-chip microfluidic device. The glomerulus-on-a-chip also mimics adriamycin-induced albuminuria and podocyte injury. This in vitro model of human glomerular function with mature human podocytes may facilitate drug development and personalized-medicine applications.

Treatment of post-burn secondary foot deformity

A case of simultaneous correction of severe post-burn deformity with limited movement in the ankle joint, subluxation and contractures in the metatarsophalangeal joints in a 3-year old child is presented. Excision of scar tissues, tendinous plasty, capsulotomy of involved joints were performed. To close the defect, a full-thickness skin graft was used. In two years, no signs of recurrence were observed.

Diabetic foot complications in a secondary foot hospital: A clinical audit.

Previous studies conducted in Australian hospital settings suggest high variability in assessments, investigations, and management of diabetic foot infections and poor adherence to widely accessible evidence-based protocols and guidelines. Diabetic foot complications require a multidisciplinary approach and often involve both medical and surgical teams during inpatient care. The aim of this clinical audit was to better understand the scope of diabetes-related foot complications, evaluate whether current assessment and management strategies are in line with best practice guidelines, and to formulate future models of care. A retrospective review of patients was carried out between 12 July 2012 and 11 July 2013. Recorded assessments of inpatient care, including risk factors, surgery, length of stay, interdepartmental referrals, and antibiotic administration were reviewed. There were 24 admissions in 12 months (total patients n=19). Fifty-eight per cent of patients were admitted to the medical ward. More than one-quarter had evidence of osteomyelitis. While one patient required intensive care unit (ICU) management, there was no inpatient mortality. Two patients experienced significant delay to undergo initial surgical intervention presumably because of failed medical treatment. Clinical data was recorded poorly, especially regarding neuropathy, HbA1c, and clinical examination findings. Twelve per cent of patients did not undergo any follow-up. The average length of stay was 12 days. One-half of the cohort was not evaluated by the endocrinology department. This audit highlights the need for improved care for patients with diabetic foot complications and better coordination among the multidisciplinary teams involved.

The Incidence of Asymptomatic Onychomycosis in Diabetes Mellitus

In people with diabetes mellitus (DM), fungal infection is believed to occur more frequently. Toenail onychomycosis (TOM), a common type of fungal infection is asymptomatic in most cases. In order to study the incidence and prevalence of previously undiagnosed TOM in a population of subjects with DM attending for routine annual foot screening, nail plate and sub-ungual tissue samples were collected for microbiological analysis. Examining clinicians also made a graded visual diagnosis of onychomycosis at the same visit. Various blood samples markers of diabetes and its associated complications were also reviewed. 88 consecutive subjects with diabetes [mean age 78.4 (± SD 10.2) and duration 7.9 (± SD 8.1) years] were recruited to the study, and 29.5% had a positive (laboratory) diagnosis of TOM. There was no correlation with the incidence of TOM and duration of diabetes, other demographic data, or various biochemical parameters, but there was a trend for association of TOM to reduced sensation in healthy people with diabetes. Clinician were making more accurate (82%) diagnosis of TOM than that of healthy nail plate (53.8%) when compared with microbiological studies. As TOM is very common and the benefit of its treatment has not been confirmed, the value of routine laboratory screening for TOM as part of annual assessment of diabetes is not established. Our study shows that TOM is very common so further studies are needed to see if screening and treatment of TOM will prevent future secondary foot and leg infection in people with diabetes.

Small molecule membrane transporters in the mammalian podocyte: a pathogenic and therapeutic target.

The intriguingly complex glomerular podocyte has been a recent object of intense study. Researchers have sought to understand its role in the pathogenesis of common proteinuric diseases such as minimal change disease and focal segmental glomerular sclerosis. In particular, considerable effort has been directed towards the anatomic and functional barrier to macromolecular filtration provided by the secondary foot processes, but little attention has been paid to the potential of podocytes to handle plasma proteins beyond the specialization of the slit diaphragm. Renal membrane transporters in the proximal tubule have been extensively studied for decades, particularly in relation to drug metabolism and elimination. Recently, uptake and efflux transporters for small organic molecules have also been found in the glomerular podocyte, and we and others have found that these transporters can engage not only common pharmaceuticals but also injurious endogenous and exogenous agents. We have also found that the activity of podocyte transporters can be manipulated to inhibit pathogen uptake and efflux. It is conceivable that podocyte transporters may play a role in disease pathogenesis and may be a target for future drug development.