Effacement Of Foot Processes Research Articles

Renal involvement in TAFRO syndrome: a review.

Renal involvement in TAFRO syndrome is characterized clinically by general edema with ascites and pleural effusions and a rapidly progressive decline in renal function, with urinary protein levels of usually less than 1g/day. The histologic features of the kidneys can be described as glomerular microangiopathy characterized by mesangiolysis or mesangial loosening, endothelial cell proliferation, edematous opening in the subendothelial space, and glomerular basement membrane (GBM) doubling due to newly formed basement membrane. Findings such as rupture of the GBM, foot-process effacement or fusion, and epithelial cell loss are rare, and thrombus formation is difficult to identify in the glomerulus. Furthermore, immunodeposits are not seen on immunofluorescence staining or electron microscopy. Unlike adults, in addition to the glomerular lesions described above, adolescents appear to show intimal proliferation of the arterioles and interlobular arteries to the vascular poles and occlusion of the vascular lumen.

Autoantibodies Targeting Proteasome Subunit Alpha Type 1 in Autoimmune Podocytopathies.

The antibody against proteasome subunit alpha type 1 (PSMA1) is a podocyte autoantibody in idiopathic nephrotic syndrome (INS) children identified in our previous study. The aim of this study was to explore the characteristics of INS in children and the mechanism underlying its involvement in the development of INS. The levels of serum anti-PSMA1 autoantibodies in children were detected via protein microarray and compared among different disease groups. The recombinant PSMA1 protein was injected subcutaneously and intraperitoneally into mice to observe glomerular morphology and function. The PSMA1-knockdown and PSMA1-overexpressing cell lines were constructed from mouse podocytes, and their cytoskeleton and function were analyzed. Homozygous zebrafish with psma1 knockout were observed. The levels of serum anti-PSMA1 autoantibodies were higher in INS children and varied with urinary protein. In mice immunized with PSMA1, the presence of serum anti-PSMA1 autoantibody caused albuminuria and damage to the glomerular filtration membrane. Deficiency of PSMA1 impaired the podocyte cytoskeleton and physiological function. Complete deletion of psma1 caused edema, abnormal glomerular morphology and effacement of foot processes in zebrafish. PSMA1 played an important role in the maintenance of podocyte morphology and function.

Computational Analysis of Boerhavia diffusa Plant Extracts Targeting Alpha actinin4 against Focal Segmental Glomerulosclerosis

The most common protein lost in urine (proteinuria) is albumin, which is a symptom of the kidney illness focal segmental glomerulosclerosis (FSGS). It causes damage to podocyte foot processes, resulting in effacement of foot processes, and injury to foot processes leads to the leaking of plasma proteins into the urine, resulting in nephrotic syndrome. Alpha actinin (ACTN4) is extensively produced in glomerular podocytes, has a function in non-muscle cytoskeletal activity, and is enhanced early in the course of nephrotic illness in various types. Mutations in ACTN4 induce autosomal and sporadic steroid-resistance nephritic syndrome, which causes disturbance in podocyte foot process and function. All of the FSGS-causing mutations are situated on actin actin-binding domain of the ACTN4 protein. FSGS is typically treated by decreasing dietary salt and using immune-suppressing medications such as glucocorticoids. There is a risk associated with these drugs for cancer patients. Several studies have found that up to 80% of individuals with primary FSGS are resistant to steroid therapy, even though all other therapeutic options have been exhausted. Patients with steroid-resistant FSGS have a higher incidence of end-stage renal failure. Therefore, several new treatment strategies were put forward to cure the disease form of which use of phytochemicals is one such sustainable modality. In addition to this, our study intended to predict the stability of mutant protein’s structure as compared to wild type structure through molecular dynamic simulation analysis. With this, we extend our study to predict the potency of phytochemicals of Boerhaviadiffusa against FSGS by using molecular docking analysis. From our study, we conclude that the actin-binding domain of ACTN4 protein becomes more unstable or loses its stability after the mutation at position W59R among all the studied mutation positions. The boeravinone F phytochemical of the Boerhaviadiffusa plant shows the best inhibitory effect on the mutant actin-binding domain of ACTN4 protein and it confirms a stable binding confirmation at minimum energy of -7.5 kcal/mol. Hence it may be taken into consideration for future research work.

Quantifiable and reproducible phenotypic assessment of a constitutive knockout mouse model for congenital nephrotic syndrome of the Finnish type

Steroid-resistant nephrotic syndrome (SRNS) is the second most frequent cause of childhood chronic kidney disease. Congenital nephrotic syndrome of the Finnish type (CNF) (MIM# 256300) is caused by biallelic variants in the gene NPHS1, encoding nephrin, an integral component of the kidney filtration barrier. No causal treatments exist, and children inevitably require kidney replacement therapy. In preparation for gene replacement therapy (GRT) in CNF, we established a quantifiable and reproducible phenotypic assessment of the nephrin-deficient CNF mouse model: 129/Sv-Nphs1tm1Rkl/J. We assessed the phenotypic spectrum of homozygous mice (Nphs1tm1Rkl/Nphs1tm1Rkl) compared to heterozygous controls (Nphs1tm1Rkl/Nphs1WT) by the following parameters: 1. cohort survival, 2. podocyte foot process (FP) density per glomerular basement membrane (GBM) using transmission electron microscopy, 3. tubular microcysts in brightfield microscopy, and 4. urinary albumin/creatinine ratios. Nphs1tm1Rkl/Nphs1tm1Rkl mice exhibited: 1. perinatal lethality with median survival of 1 day, 2. FP effacement with median FP density of 1.00 FP/µm GBM (2.12 FP/µm in controls), 3. tubular dilation with 65 microcysts per section (6.5 in controls), and 4. increased albumin/creatinine ratio of 238 g/g (4.1 g/g in controls). We here established four quantifiable phenotyping features of a CNF mouse model to facilitate future GRT studies by enabling sensitive detection of phenotypic improvements.

#2518 The decrease of the filtration slit density, not proteinuria, is the early indication of the filtration barrier dysfunction

Abstract Background and Aims Intact podocyte foot processes (FP) play a crucial role in ensuring proper selective filtration in the kidney. Changes in FP morphology are thought to be the initial trigger for proteinuria, but definitive proof is still missing. As FP structures are below the optical resolution of light microscopes, changes are investigated by electron microscopy (EM) on ultrathin sections. In this study, the correlation between morphology and proteinuria was investigated using the Podocyte Exact Morphology Measurement Procedure (PEMP), a 3D-SIM-based technique. Our investigation was focused on podocyte damage caused by treatment with nephrotoxic serum (NTS) as a model for focal segmental glomerulosclerosis. Method The mice were treated with nephrotoxic serum (NTS), and kidneys were taken at 24, 48, and 72 hours after injection. Kidney sections (3 µm thick) were prepared for 3D-SIM analysis after staining with podocin and integrin and then fixed in formalin and embedded in paraffin. Filtration slit density (FSD) was quantified using the Podocyte Exact Morphology Measurement Procedure (PEMP) as described earlier (Siegerist et al. 2017). In addition, EM was performed on glutaraldehyde-fixed tissue samples using standard protocols. The albumin to creatinine ratio was also determined. Results We observed that NTS-treated mice developed moderate proteinuria after 48 hours. Using the super-resolution microscopy-based 3D-SIM technique to quantify podocyte PF morphology by calculating the filtrations slit density (FSD) revealed a significant reduction of the FSD in NTS-treated mice already after 24 hours. This structural change was confirmed by transmission electron microscopy analysis, showing that the effacement of podocytes FPs is the first sign of a change in the filtration barrier function before proteinuria is measurable. Conclusion PEMP analysis of standard histological sections from NTS-injected and control mice demonstrated that the first event in the functional loss of the size selectivity of the filtration barrier is the effacement of FP morphology, followed by detectable proteinuria. Therefore, the assessment of the FSD is a promising strategy for early and accurate detection of the loss of filtration barrier integrity, providing opportunities for faster and more precise diagnostics.

#2862 Genetics and electron microscopy: complementary tools for management of focal segmental glomerulosclerosis?

Abstract Background and Aims Focal segmental glomerular sclerosis (FSGS) defines a histologic lesion that represents a pattern of injury associated to different pathologic conditions affecting both children and adults. It is classified into primary forms, presumably immune-mediated, secondary forms, determined by a maladaptive response to different conditions, genetic and undetermined forms. According to Mayo Clinic classification, primary and secondary forms can de differentiated using electron microscopy (EM), with a podocytes foot process effacement (FPE) ≥80% identifying primary forms. Better understanding the role of Genetics in FSGS and response to treatment in different forms is of vital importance in this rare and multifaceted condition. We analyzed a discrete cohort of patients with FSGS examined both with EM and genetic testing (GT). Main aims of our study were: 1) assessment of GT in primary and secondary forms; 2) analysis of response to therapy according to histologic and genetic classification. Method This is a retrospective observational study on patients that received a histological diagnosis of FSGS, including EM, and underwent NGS genetic testing from January 2020 to June 2023. Primary and secondary forms of FSGS were distinguished by degree of pedicle fusion at EM, according to Mayo Clinic classification. Clinical, laboratory, genetic data and used therapies were recorded. Complete response (CR) to therapy was defined as a reduction of proteinuria to 0.3 g/day, with normalization of albuminemia. Partial response (PR) was defined as halving proteinuria into a sub-nephrotic range and increasing albuminemia up to 3.5 g/dL. Absent response was defined as a persisting nephrotic range proteinuria. Results In the study period, 33 patients receiving a histologic diagnosis of FSGS with EM examination underwent NGS testing. Of these, 25 had available genetic results and were included. Ten patients (40%) were females, 15 (60%) were males; mean age was 40.8 years (SD ±14.5); 20 (80%) were Caucasian, 4 (16%) Black, 1 (4%) Asian. Based on EM, 11 (44%) were classified as primary forms, 14 (56%) as secondary forms. Twelve (48%) patients presented with nephrotic syndrome. Of these, 10 had a primary form. Seven (28%) had nephrotic-range proteinuria without hypoalbuminemia, 6 (24%) had subnephrotic range proteinuria. Six (54.5%) of the primary forms and 8 (57.1%) of the secondary forms had a positive genetic testing (p = 0.78). Among secondary forms, through GT, one diagnosis of Fabry disease and one of Tuberous Sclerosis were made in patients without clear systemic symptoms. Fabry disease was confirmed by presence of zebra bodies at EM. All primary forms received immunosuppressive therapies, all being rituximab-based regimens; all secondary forms received support therapy. Nine (81.1%) primary forms and 9 (64.3%) secondary forms had at least a PR to treatment (4 CR in primary group and 1 CR in secondary group) (p = 0.33). Among primary forms, 5 of 6 patients (83%) with a positive GT had at least a PR with a strong immunosuppressive therapy. In secondary forms group 4 of 8 patients (50%) had at least PR with offered support therapy. Fabry and Tuberous Sclerosis were offered specific treatments with benefit. Conclusion Percentage of positive GT did not differ in primary and secondary forms of FSGS as defined by Mayo Clinic classification. Thus a positive GT should not be considered a unique feature of secondary forms. GT was critical for an unexpected diagnosis of systemic hereditary diseases, thus enabling adequate target therapies. We believe GT should be offered to all patients with FSGS. In primary FSGS, defined according to EM, more than 80% of patients had at least a PR following immunosuppressive therapy, even in patients with positive GT. In conclusion, as clinical presentation and light microscopy are not sufficient for an accurate diagnosis, combining EM and GT is of utmost importance in proper classification and management of FSGS.

#1639 Nephrotic syndrome with anti-nephrin antibodies—case report of minimal change glomerulonephritis

Abstract Background and Aims Minimal change disease (MCD) in adults is one of the causes of nephrotic syndrome. Recently, anti-nephrin antibodies localized in glomerular podocytes have been described in a certain subset of patients with MCD, supporting the autoimmune etiology of the disease. Method A 79-year-old man with arterial hypertension, hypothirosis and mild dementia presented with new-onset nephrotic syndrome. Peripheral and periocular edema, hypoalbuminemia (serum albumin 17 g/l), proteinuria 15 g/day and hypercholesterolemia (serum cholesterol 6.7 mmol/l) were noted. Blood pressure was 133/85 mmHg. Creatinine was 165 mcmol/l on admission and increased to 361 mcmol/l in the following days. Immunologic tests were negative, including antibodies against phospholipase A2-receptor (anti-PLA2R) and thrombospondin-7A (anti-THSD7A). Anti-HBc was positive, HBV DNA was below 15 IU/ml. Liver tests and abdominal ultrasound were normal, with no signs of liver disease. Results Renal biopsy revealed 18 glomeruli with global sclerosis of one glomerulus (1/18) and 5% interstitial fibrosis. Tubular damage was noted, which could be attributed to severe proteinuria. Immunofluorescence showed discrete IgG podocyte deposits with a uniform kappa/lambda distribution and a high probability of idiopathic minimal change disease due to anti-nephrin antibodies. Electron microscopy showed diffuse effacement of the podocyte foot processes without deposits. The serum taken after the biopsy and before the start of immunosuppressive therapy was positive for anti-nephrin antibodies. Due to a possible pharmacokinetic interaction with tenofovir, which the patient was receiving because of his anti-HBc positivity, we decided not to use cyclosporine. In addition to standard therapy with angiotensin convertase inhibitors, calcium channel blockers and furosemide, the patient was treated with low-dose oral glucocorticoids and mycophenolic acid. In addition, he received rituximab. After a few weeks, the oral glucocorticoids and mycophenolic acid were discontinued. The nephrotic syndrome regressed completely and renal function improved. Conclusion Anti-nephrin antibodies have contributed to a subgroup of patients with MCD, they can be found in a renal biopsy and are also detected in the serum of these patients. Targeted anti-B-cell therapy should be introduced to improve the prognosis of patients.

Zyxin is important for the stability and function of podocytes, especially during mechanical stretch

Podocyte detachment due to mechanical stress is a common issue in hypertension-induced kidney disease. This study highlights the role of zyxin for podocyte stability and function. We have found that zyxin is significantly up-regulated in podocytes after mechanical stretch and relocalizes from focal adhesions to actin filaments. In zyxin knockout podocytes, we found that the loss of zyxin reduced the expression of vinculin and VASP as well as the expression of matrix proteins, such as fibronectin. This suggests that zyxin is a central player in the translation of mechanical forces in podocytes. In vivo, zyxin is highly up-regulated in patients suffering from diabetic nephropathy and in hypertensive DOCA-salt treated mice. Furthermore, zyxin loss in mice resulted in proteinuria and effacement of podocyte foot processes that was measured by super resolution microscopy. This highlights the essential role of zyxin for podocyte maintenance in vitro and in vivo, especially under mechanical stretch.

CD36-mediated podocyte lipotoxicity promotes foot process effacement.

Lipid metabolism disorders lead to lipotoxicity. The hyperlipidemia-induced early stage of renal injury mainly manifests as podocyte damage. CD36 mediates fatty acid uptake and the subsequent accumulation of toxic lipid metabolites, resulting in podocyte lipotoxicity. Male Sprague-Dawley rats were divided into two groups: the normal control group and the high-fat diet group (HFD). Podocytes were cultured and treated with palmitic acid (PA) and sulfo-N-succinimidyl oleate (SSO). Protein expression was measured by immunofluorescence and western blot analysis. Boron-dipyrromethene staining and Oil Red O staining was used to analyze fatty acid accumulation. Podocyte foot process (FP) effacement and marked proteinuria occurred in the HFD group. CD36 protein expression was upregulated in the HFD group and in PA-treated podocytes. PA-treated podocytes showed increased fatty acid accumulation, reactive oxygen species (ROS) production, and actin cytoskeleton rearrangement. However, pretreatment with the CD36 inhibitor SSO decreased lipid accumulation and ROS production and alleviated actin cytoskeleton rearrangement in podocytes. The antioxidant N-acetylcysteine suppressed PA-induced podocyte FP effacement and ROS generation. CD36 participated in fatty acid-induced FP effacement in podocytes via oxidative stress, and CD36 inhibitors may be helpful for early treatment of kidney injury.

Obesity-related glomerulopathy is associated with elevated WT1 expression in podocytes.

The prevalence of obesity is increasing worldwide at an alarming rate. In addition to the increased incidence of cardiovascular and metabolic diseases, obesity is the most potent risk factor for developing chronic kidney disease (CKD). Although systemic events such as hemodynamic factors, metabolic effects, and lipotoxicity were implicated in the pathophysiology of obesity-related glomerulopathy (ORG) and kidney dysfunction, the precise mechanisms underlying the association between obesity and CKD remain unexplored. In this study, we employed spontaneous WNIN/Ob rats to investigate the molecular events that promote ORG. Further, we fed a high-fat diet to mice and analyzed the incidence of ORG. Kidney functional parameters, micro-anatomical manifestations, and podocyte morphology were investigated in both experimental animal models. Gene expression analysis in the rodents was compared with human subjects by data mining using Nephroseq and Kidney Precision Medicine Project database. WNIN/Ob rats were presented with proteinuria and several glomerular deformities, such as adaptive glomerulosclerosis, decreased expression of podocyte-specific markers, and effacement of podocyte foot process. Similarly, high-fat-fed mice also showed glomerular injury and proteinuria. Both experimental animal models showed increased expression of podocyte-specific transcription factor WT1. The altered expression of putative targets of WT1 such as E-cadherin, podocin (reduced), and α-SMA (increased) suggests elevated expression of WT1 in podocytes elicits mesenchymal phenotype. Curated data from CKD patients revealed increased expression of WT1 in the podocytes and its precursors, parietal epithelial cells. WT1 is crucial during nephron development and has minimal expression in adult podocytes. Our study discovered elevated expression of WT1 in podocytes in obesity settings. Our analysis suggests a novel function for WT1 in the pathogenesis of ORG; however, the precise mechanism of WT1 induction and its involvement in podocyte pathobiology needs further investigation.

Loss of MTX2 causes mitochondrial dysfunction, podocyte injury, nephrotic proteinuria and glomerulopathy in mice and patients.

Proteinuria is a common and important clinical manifestation of chronic kidney disease (CKD) and an independent risk factor for the progression of kidney disease. As a component of the glomerular filtration barrier (GFB), podocyte plays a key role in the pathogenesis of glomerular diseases and proteinuria. However, the pathophysiology of glomerular diseases associated with mitochondrial function is incompletely understood. Here, we identified three novel mutations in MTX2, encoding a membrane protein in mitochondria, associated with multisystem manifestations including nephrotic proteinuria and kidney injury in two Chinese patients. Conditional podocyte-specific Mtx2 knockout (Pod-Mtx2-KO) mice present a series of podocyte and glomerular abnormalities from 8 weeks to old age, including microalbuminuria, glomerular mesangial hyperplasia, fusion and effacement of foot process. MTX2 deficiency impaired podocyte functions in vitro, manifested by reductions of adhesion, migration and endocytosis, which were further restored by overexpression of MTX2. Moreover, MTX2 defects led to abnormal mitochondrial structure and dysfunction, evidenced with defects of complex I and III, increased production of reactive oxygen species (ROS), and decreased protein levels of Sam50-CHCHD3-Mitofilin axis in the mitochondrial intermembrane space bridging (MIB) complex which is responsible for maintaining mitochondrial cristae morphology. Collectively, these findings reveal that the normal expression of MTX2 in glomerulus plays an important role in the adhesion, migration, endocytosis, proliferation and other physiological functions of podocytes, which may be realized by maintaining the morphological structure and function of mitochondria. Abnormal expression of MTX2 can lead to mitochondrial dysfunction and structural abnormalities by Sam50-CHCHD3-Mitofilin axis in podocyte, which further induces podocyte injury, glomerular lesions and proteinuria.

Cyclosporine A Protects Podocytes by Regulating Transgelin in Adriamycin-Induced Podocyte Injury.

The calcineurin inhibitor cyclosporine A (CsA) has been shown to effectively reduce proteinuria. However, its precise mechanism is still not fully understood. Our previous study showed that CsA reduced proteinuria by directly stabilizing the foot process (FP) cytoskeletal structure via cofilin-1, suggesting that synaptopodin, a podocyte-specific actin protein, is not the sole target of CsA in podocytes. In this study, we established an adriamycin (ADR)-induced nephropathy rat model and a cultured podocyte injury model. We employed Western blotting and immunofluorescence techniques to assess the expression and distribution of transgelin, Krüppel-like factor-4 (KLF-4), nephrin, and synaptopodin. We observed a significant increase in proteinuria levels accompanied by loss of normal FP structure in the ADR-induced nephropathy rat model. The levels of the actin cross-linking protein transgelin were increased significantly, while those of the podocyte-specific molecules nephrin and synaptopodin were decreased in vivo. Treatment with CsA effectively reduced proteinuria while restoring FP effacement stability in ADR-induced nephropathy models and restoring the expression of transgelin, nephrin, and synaptopodin both in vivo and in vitro. Furthermore, CsA treatment dose-dependently decreased transgelin levels while significantly increasing KLF-4 expression in injured podocytes. In addition, CsA failed to downregulate transgelin when KLF-4 was specifically knocked down. Our findings suggest that CsA protects against podocyte injury by downregulating abnormally high levels of transgelin via upregulation of KLF-4 expression.

Anti-Glomerular Basement Membrane Disease in a Six-year-old Child: A Rare Presentation

Antiglomerular basement membrane (anti-GBM) disease is a rare small vessel vasculitis caused by auto-antibodies targeting auto-antigen expressed in basement membranes of capillary beds in lungs and kidneys. A six-year-old male presented with fever, cough, and generalized weakness for three days with respiratory distress for one day. The child had three prior admissions with similar complaints and was treated as a case of lower respiratory tract infection with severe iron deficiency anemia in the past. Multiple packed red cell transfusions were required in these past admissions. In this current admission, he had tachycardia, tachypnea, severe pallor, right-sided lung crepitations & bronchial breath sounds. The hemogram showed severe anemia and leukocytosis. Urine examination showed mild proteinuria and microscopic hematuria. Chest X-ray (current admission) showed right middle and lower zone consolidation. HRCT of the chest revealed an interstitial pattern. A lung biopsy showed pulmonary hemorrhage and positive Prussian blue staining. Raised anti-GBM titers confirmed diagnosis of anti-GBM disease. Renal biopsy revealed normal glomerular morphology on histopathology; with immune fluorescence showing linear positivity for IgG (3+), Kappa (3+), and Lambda (3+). Electron microscopy reported mild effacement of visceral epithelial foot processes and subepithelial/intramembranous electron-dense deposits. He was treated with intravenous methylprednisone for 5 days followed by oral prednisolone for one month, which was thereafter tapered to a lower maintenance dose for 18 months. The child received six cycles of intravenous cyclophosphamide along with a daily low dose of steroids. Currently, the child is asymptomatic after one year of follow-up with seroconversion (antiGBM titers decreased) and normalization of the chest radiograph.

#3626 A CASE OF PODOCYTE DISEASE WITH AZOTEMIA FOLLOWING TREATMENT WITH INTRAVENOUS IBANDRONATE

Abstract Background and Aims Bisphosphonate use may induce several types of nephrotoxicity. There are many reports on pamidronate-associated glomerular disease, such as focal segmental glomerulosclerosis and zoledronate-associated acute tubular necrosis. However, there are limited reports on ibandronate-associated nephrotoxicity. Herein, we describe a case of podocyte disease accompanied with azotemia following intravenous administration of ibandronate. Method Case report Results An 88-year-old female was referred to the emergency room due to grade 4 edema that developed 1 month before. The patient was diagnosed with osteoporosis 20 months earlier, for which she received quarterly intravenous administration of ibandronate (3 mg). The last dose was administered 2 weeks ago. The patient had no history of diabetes, hypertension, or chronic kidney disease. Laboratory tests revealed heavy proteinuria with a protein/creatinine ratio (PCR) of 32 and hypoalbuminemia (1.9 g/dL) with elevated serum creatinine (sCr) (1.8 mg/dL). Values of serum complement and electrophoresis were within normal ranges. The renal ultrasonogram was unremarkable. A renal biopsy was performed and light microscopy showed mild mesangial hypercellularity and segmental amorphous collagenous deposition in the glomerulus (Fig. 1-A). The tubules revealed focal marked atrophy and interstitial fibrosis (Fig. 1-B). Immunofluorescence studies were unremarkable. Electron microscopy showed diffuse effacement of foot processes and no electron-dense deposit (Fig. 1-C), consistent with podocyte disease favoring focal segmental glomerulosclerosis. The patient was treated with tacrolimus and fimasartan. Steroid use was spared owing to severe osteoporosis. Three months later, the edema subsided and laboratory findings improved (PCR 0.64, serum albumin 4 g/dL, sCr 1.23 mg/dL). Conclusion Compared to pamidronate and zoledronate, ibandronate is more highly protein-bound with a significantly shorter renal tissue half life, which might explain the rarity of ibandronate-related nephrotoxicity. Contrary to the trend of other bisphosphonates nephropathies, our case showed simultaneous podocyte disease and tubule damage. We recommend close monitoring of proteinuria and renal function for prompt nephrotoxicity detection in patients treated with ibandronate.

Wen-Shen-Jian-Pi-Hua-Tan decoction protects against early obesity-related glomerulopathy by improving renal bile acid composition and suppressing lipogenesis, inflammation, and fibrosis

BackgroundObesity is an independent predictor of chronic kidney disease (CKD) development and may directly lead to kidney lesions such as obesity-related glomerulopathy (ORG) which might play a vital pathogenic role in obese patients with CKD. Wen-Shen-Jian-Pi-Hua-Tan decoction (WSHT) has been clinically used for the treatment of obesity and obesity-related metabolic diseases for years. However, the renoprotective effects and potential mechanism of action of WSHT against ORG remain unknown. PurposeThis study aimed to explore the potential effect of WSHT on ORG and reveal its mechanisms in high-fat diet (HFD)-induced obese rats. MethodsAn animal model of early stage ORG was established using HFD-induced obese rats. After treatment with WSHT for 6 weeks, an integrated metabolomics and molecular biology strategy was utilized to illustrate the effects and mechanism of WSHT on ORG. First, UPLC-ESI-MS/MS-based targeted metabolomics was used to analyze renal bile acid (BA) levels. Biochemical, histological, and immunofluorescence assays; electron microscopy; and western blotting were performed to evaluate the efficacy of WSHT against ORG and its underlying mechanisms in vivo. ResultsOur results showed that an HFD led to hyperlipidemia, proteinuria, renal lipid deposition, effacement of podocyte foot processes, and increased expression of proinflammatory factors and profibrotic growth factors in ORG rats. In addition, an HFD decreased the levels of renal BAs such as cholic acid, chenodeoxycholic acid, and lithocholic acid. After 6 weeks of treatment, WSHT markedly attenuated dyslipidemia and reduced body, kidney and epididymal fat weights in ORG rats. WSHT also significantly increased BA levels, suggesting that it altered BA composition; the effects of BAs are closely associated with farnesoid X receptor (FXR) activation. WSHT alleviated fat accumulation, podocyte loss and proteinuria, and reduced the expression of proinflammatory cytokines and profibrotic growth factors in the kidneys of ORG rats. Finally, WSHT remarkably upregulated the renal expression of FXR and salt-induced kinase 1 and blocked the renal expression of sterol regulatory element–binding protein-1c and its target genes. ConclusionWSHT attenuated early renal lesions in ORG rats by improving renal BA composition and suppressing lipogenesis, inflammation and fibrosis. This study develops a new way to alleviate obesity-induced renal damages.

Proteins Secreted by Lung Cancer Cells Induce the Onset of Proteinuria via Focal Adhesion Kinase Signaling in Mice

Paraneoplastic nephrotic syndrome (PNS) is a complication seen in cancer patients. Ultrastructural examination shows the accumulation of proteins and the presence of foot process (FP) effacement in the glomeruli of PNS patients. Previously, we reported that orthotopic xenografts of Lewis lung carcinoma 1 in C57BL/6 mice caused them to develop lung cancer with albuminuria. This implies that these mice can be used as a model of human disease and suggests that Lewis lung carcinoma 1 cell–secreted proteins (LCSePs) contain nephrotoxic molecules and cause inflammation in renal cells. As podocyte effacement was present in glomeruli in this model, such podocyte injury may be attributable to either soluble LCSeP or LCSeP deposits triggering pathological progression. LCSePs in conditioned media was concentrated for nephrotoxicity testing. Integrin-focal adhesion kinase (FAK) signaling and inflammatory responses were evaluated in podocytes either exposed to soluble LCSePs or seeded onto substrates with immobilized LCSePs. FAK phosphorylation and interleukin-6 expression were higher in podocytes attached to LCSePs substrates than in those exposed to soluble LCSePs. Notably, LCSeP-based haptotaxis gave rise to altered signaling in podocytes. When podocytes were stimulated by immobilized LCSePs, FAK accumulated at focal adhesions, synaptopodin dissociated from F-actin, and disrupting the interactions between synaptopodin and α-actinin was observed. When FAK was inhibited by PF-573228 in immobilized LCSePs, the association between synaptopodin and α-actinin was observed in the podocytes. The association of synaptopodin and α-actinin with F-actin allowed FP stretching, establishing a functional glomerular filtration barrier. Therefore, in this mouse model of lung cancer, FAK signaling prompts podocyte FP effacement and proteinuria, indicative of PNS.

Kidney injury molecule-1 and podocalyxin dysregulation in an arginine vasopressin induced rodent model of preeclampsia

ObjectiveTo assess renal injury in an arginine vasopressin (AVP) rodent model of preeclampsia. Study designUrinary expression of kidney injury molecule-1 (KIM-1), urinary protein and creatinine was determined in rodents (n = 24; pregnant AVP, pregnant saline, non-pregnant AVP and non-pregnant saline), which received a continuous dose of either AVP or saline via subcutaneous mini osmotic pumps for 18 days, using a Multiplex kidney toxicity immunoassay. Renal morphology was assessed using haematoxylin and eosin staining and transmission electron microscopy. The immunolocalization of KIM-1 and podocalyxin was qualitatively evaluated using immunohistochemistry. ResultsUrinary KIM-1 and urinary protein levels were significantly increased in treated vs. untreated rats on gestational days 8 (p < 0.05), 14 (p < 0.001) and 18 (p < 0.001). The pregnant rats displayed a lower trend of creatinine compared to the non-pregnant groups, albeit non-significantly. KIM-1 was immunolocalized in the proximal convoluted tubules in AVP treated vs. untreated groups. In contrast, podocalyxin was weakly immunostained within glomeruli of pregnant AVP treated vs. pregnant untreated rats. Histological evaluation revealed reduced Bowman’s space, with some tubular and blood vessel necrosis in the pregnant treated group. Ultrastructural observations included effacement and fusion of podocyte foot processes, glomerular basement membrane abnormalities, podocyte nuclear crenations, mitochondrial oedema and cristae degeneration with cytoplasmic lysis within treated tissue. ConclusionOur findings demonstrate region-specific kidney injury particularly glomerular impairment and endothelial injury in AVP-treated rats. The findings highlight the utility of this model in studying the mechanisms driving renal damage in a rodent model of preeclampsia.

Proteinuria in thrombotic microangiopathy is associated with partial podocytopathy

ABSTRACT Background. Thrombotic microangiopathy (TMA) results in acute kidney injury, but the cause of heavy proteinuria in this disorder is puzzling. The goal of this study was to determine if there were significant effacement of foot processes and CD133-positive hyperplastic podocytes in TMA to explain the proteinuria. Methods. The study included 12 negative controls (renal parenchyma removed from renal cell carcinoma) and 28 thrombotic microangiopathy due to different etiologies. The percent of foot process effacement was estimated, and proteinuria level was obtained for each TMA case. Both groups of cases were stained for CD133 by immunohistochemical method, and the number of positive CD133 in hyperplastic podocytes was counted and analyzed. Results. Nineteen (19) of 28 (68%) TMA cases had nephrotic range proteinuria (urine protein/creatinine >3). Twenty-one (21) of 28 (75%) TMA cases showed positive CD133 staining in scattered hyperplastic podocytes within Bowman’s space but was absent in control cases. The percent of foot process effacement (56 ± 4%) correlated with proteinuria (protein/creatinine ratio 4.4 ± 0.6) (r = 0.46, p = .0237) in TMA group. Conclusion. Our data indicate that the proteinuria in TMA can be associated with significant effacement of foot processes. CD133-positive hyperplastic podocytes can be seen in the majority of TMA cases of this cohort, indicating a partial podocytopathy.

Podocyte Geranylgeranyl Transferase Type-I Is Essential for Maintenance of the Glomerular Filtration Barrier.

A tightly regulated actin cytoskeleton attained through balanced activity of RhoGTPases is crucial to maintaining podocyte function. However, how RhoGTPases are regulated by geranylgeranylation, a post-translational modification, has been unexplored. The authors found that loss of the geranylgeranylation enzyme geranylgeranyl transferase type-I (GGTase-I) in podocytes led to progressive albuminuria and foot process effacement in podocyte-specific GGTase-I knockout mice. In cultured podocytes, the absence of geranylgeranylation resulted in altered activity of its downstream substrates Rac1, RhoA, Cdc42, and Rap1, leading to alterations of β1-integrins and actin cytoskeleton structural changes. These findings highlight the importance of geranylgeranylation in the dynamic management of RhoGTPases and Rap1 to control podocyte function, providing new knowledge about podocyte biology and glomerular filtration barrier function. Impairment of the glomerular filtration barrier is in part attributed to podocyte foot process effacement (FPE), entailing disruption of the actin cytoskeleton and the slit diaphragm. Maintenance of the actin cytoskeleton, which contains a complex signaling network through its connections to slit diaphragm and focal adhesion proteins, is thus considered crucial to preserving podocyte structure and function. A dynamic yet tightly regulated cytoskeleton is attained through balanced activity of RhoGTPases. Most RhoGTPases are post-translationally modified by the enzyme geranylgeranyl transferase type-I (GGTase-I). Although geranylgeranylation has been shown to regulate activities of RhoGTPases and RasGTPase Rap1, its significance in podocytes is unknown. We used immunofluorescence to localize GGTase-I, which was expressed mainly by podocytes in the glomeruli. To define geranylgeranylation's role in podocytes, we generated podocyte-specific GGTase-I knockout mice. We used transmission electron microscopy to evaluate FPE and measurements of urinary albumin excretion to analyze filtration barrier function. Geranylgeranylation's effects on RhoGTPases and Rap1 function were studied in vitro by knockdown or inhibition of GGTase-I. We used immunocytochemistry to study structural modifications of the actin cytoskeleton and β1 integrins. Depletion of GGTase-I in podocytes in vivo resulted in FPE and concomitant early-onset progressive albuminuria. A reduction of GGTase-I activity in cultured podocytes disrupted RhoGTPase balance by markedly increasing activity of RhoA, Rac1, and Cdc42 together with Rap1, resulting in dysregulation of the actin cytoskeleton and altered distribution of β1 integrins. These findings indicate that geranylgeranylation is of crucial importance for the maintenance of the delicate equilibrium of RhoGTPases and Rap1 in podocytes and consequently for the maintenance of glomerular integrity and function.

Pathological characteristics of light chain crystalline podocytopathy

Monoclonal immunoglobulin light chain (LC) crystalline inclusions within podocytes are rare, poorly characterized entities. To provide more insight, we now present the first clinicopathologic series of LC crystalline podocytopathy (LCCP) encompassing 25 patients (68% male, median age 56 years). Most (80%) patients presented with proteinuria and chronic kidney disease, with nephrotic syndrome in 28%. Crystalline keratopathy and Fanconi syndrome were present in 22% and 10%, respectively. The hematologic condition was monoclonal gammopathy of renal significance (MGRS) in 55% and multiple myeloma in 45%. The serum monoclonal immunoglobulin was IgG κappa in 86%. Histologically, 60% exhibited focal segmental glomerulosclerosis (FSGS), often collapsing. Ultrastructurally, podocyte LC crystals were numerous with variable effacement of foot processes. Crystals were also present in proximal tubular cells as light chain proximal tubulopathy (LCPT) in 80% and in interstitial histiocytes in 36%. Significantly, frozen-section immunofluorescence failed to reveal the LC composition of crystals in 88%, requiring paraffin-immunofluorescence or immunohistochemistry, with identification of kappa LC in 87%. The LC variable region gene segment, determined by mass spectrometry of glomeruli or bone marrow plasma cell sequencing, was IGKV1-33 in four and IGKV3-20 in one. Among 21 patients who received anti-plasma cell-directed chemotherapy, 50% achieved a kidney response, which depended on a deep hematologic response. After a median follow-up of 36 months, 26% progressed to kidney failure and 17% died. The mean kidney failure-free survival was 57.6 months and was worse in those with FSGS. In sum, LCCP is rare, mostly associates with IgG κappa MGRS, and frequently has concurrent LCPT, although Fanconi syndrome is uncommon. Paraffin-immunofluorescence and electron microscopy are essential to prevent misdiagnosis as primary FSGS since kidney survival depends on early diagnosis and subsequent clone-directed therapy.