Search

BACKGROUND AND AIMS Current guidelines do not clarify whether older patients with advanced chronic kidney disease may benefit of low protein diet if they are at risk of malnutrition. We compared the effects of normo-calorie/normo-protein (NP) and normo-calorie/low protein diet (LP) on nutritional status and metabolic complications related to the progression of kidney damage in these patients. METHOD This pilot study had an open label randomized-controlled design (ClinicalTrials.gov Id: NCT05015647). Thirty-five patients were treated for six months with two different diets (LP = 17) and (NP = 18). Malnutrition was assessed by Malnutrition Inflammation Score and International Society of Renal Nutrition and Metabolism criteria. Renal function was assessed by creatinine and cystatin C based eGFR. RESULTS At the end of the study, Malnutrition Inflammation Score was improved in both LP and NP groups (respectively: 3 ± 3 versus 6 ± 1.5, P = 0.020 and 3 ± 2.5 versus 6 ± 2, P = 0.012), and prevalence of protein energy wasting syndrome decreased only in LP. LP group had higher eGFRcys-C (17 ± 6 versus 12 ± 4 mL/min/1.73 m2; P CONCLUSION LP diet has a favorable impact on nutritional status as much as NP diet with possible greater benefits on the progression of kidney disease and some of its metabolic complications.

In 32-kidney transplanted patients (KTxps), the safety and the effects on BMD and mineral metabolism (MM) of one-year treatment with denosumab (DB) were studied. Femoral and vertebral BMD and T-score, FRAX score and vertebral fractures (sVF) before (T0) and after 12 months (T12) of treatment were measured. MM, renal parameters, hypocalcemic episodes (HpCa), urinary tract infections (UTI), major graft and KTxps outcomes were monitored. The cohort was composed mainly of females, n = 21. We had 29 KTxps on steroid therapy and 22 KTxps on vitamin D supplementation. At T0, 25 and 7 KTxps had femoral osteoporosis (F-OPS) and osteopenia (F-OPS), respectively. Twenty-three and six KTxps had vertebral osteoporosis (V-OPS) and osteopenia (V-OPS), respectively. Seventeen KTxps had sVF. At T12, T-score increased at femoral and vertebral sites (p = 0.05, p = 0.008). The prevalence of F-OPS and V-OPS reduced from 78% to 69% and from 72% to 50%, respectively. Twenty-five KTxps ameliorated FRAX score and two KTxps had novel sVF. At T12, a slight reduction of Ca was present, without HpCa. Four KTxps had UTI. No graft rejections, loss of graft or deaths were reported. Our preliminary results show a good efficacy and safety of DB in KTxps. Longer and randomized studies involving more KTxps might elucidate the possible primary role of DB in the treatment of bone disorders in KTxps.

7 figures.-- Supplementary material available on line (6 figures).-- This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder., Carbon nanomaterials offer excellent prospects as therapeutic agents, and among them, graphene quantum dots (GQDs) have gained considerable interest thanks to their aqueous solubility and intrinsic fluorescence, which enable their possible use in theranostic approaches, if their biocompatibility and favorable pharmacokinetic are confirmed. We prepared ultra-small GQDs using an alternative, reproducible, top-down synthesis starting from graphene oxide with a nearly 100% conversion. The materials were tested to assess their safety, demonstrating good biocompatibility and ability in passing the ultrafiltration barrier using an in vitro model. This leads to renal excretion without affecting the kidneys. Moreover, we studied the GQDs in vivo biodistribution confirming their efficient renal clearance, and we demonstrated that the internalization mechanism into podocytes is caveolae-mediated. Therefore, considering the reported characteristics, it appears possible to vehiculate compounds to kidneys by means of GQDs, overcoming problems related to lysosomal degradation., J. M. G.-D. acknowledges Spanish Ministry of Science, Innovation and Universities for his Juan de la Cierva Incorporación research contract (No. IJCI-2016-27789). This project has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie Grant Agreement No. 734834 (INFUSION) and No. 734381 (CARBO-IMmap), and from MIUR. ICN2 is supported by the Severo Ochoa program from Spanish MINECO (No. SEV-2017-0706)., Funding note : Open Access funding provided by Università degli Studi di Trieste within the CRUICARE Agreement.

Background/Aims The research of genes implicated in kidney glomerular function, eliciting cell fate program, is always at the forefront in nephrological studies. Several neurological molecules have been recently the object of study not only for their involvement in the central nervous system differentiation but also for their importance in the functionality of other organs and for mature phenotype, as in kidney. NeuroD, in CNS, is related to two functional roles, the early survival and the differentiation. The aim of our study was to ascertain the presence of NeuroD transcription factor in glomeruli and to understand which targets and mechanisms NeuroD controls. Methods: We used immunofluorescence (IF) studies on both human and mice renal tissues and on cultured podocytes to describe NeuroD distribution; then we investigated NeuroD binding to the nephrin promoter region in cultured podocytes by chromatin-immuno-precipitation (ChIP) assay. The overexpression of NeuroD in podocytes was used to establish first its role in nephrin synthesis, evaluated by real-time quantitative (RTq) PCR and western-blot (WB) and successively to determine the recovery of cell morphology after adriamycin injury, measuring foot processes length. Results: We identified NeuroD transcription factor in glomeruli, in the same cells positive for WT1 and synaptopodin, namely podocytes; subsequently we observed a differentiation dependent NeuroD distribution in cultured podocytes, and a consistent link of NeuroD with the Nephrin promoter leading to the regulation of Nephrin translation and transcription. Our data also describes NeuroD expression in cytoplasm as phosphoprotein linked to nephrin and actinin4. Preliminary experiments seem to indicate NeuroD involved in dynamics of cell shape regulation after adriamycin injury. Conclusion: we propose that NeuroD possess in podocytes a dual ability acting in the nucleus as a transcription factor and in cytoplasm stabilizing cell shape.

The research of novel markers in urinary samples, for the description of renal damage, is of high interest, and several works demonstrated the value of urinary mRNA quantification for the search of events related to renal disease or affecting the outcome of transplant kidneys. In the present pilot study, a comparison of the urine mRNA expression of specific podocyte markers among patients who had undergone clinical indication to renal transplanted (RTx, n = 20) and native (N, n = 18) renal biopsy was performed. The aim of this work was to identify genes involved in podocytes signaling and cytoskeletal regulation (NPHS1, NPHS2, SYNPO, WT1, TRPC6, GRM1, and NEUROD) in respect to glomerular pathology. We considered some genes relevant for podocytes signaling and for the function of the glomerular filter applying an alternative normalization approach. Our results demonstrate the WT1 urinary mRNA increases in both groups and it is helpful for podocyte normalization. Furthermore, an increase in the expression of TRPC6 after all kinds of normalizations was observed. According to our data, WT1 normalization might be considered an alternative approach to correct the expression of urinary mRNA. In addition, our study underlines the importance of slit diaphragm proteins involved in calcium disequilibrium, such as TRPC6.

Background: Sarcopenia is a prevalent condition in chronic kidney disease (CKD). We determined gut microbiota (gMB) composition in CKD patients with or without sarcopenia. Furthermore, we investigated whether in these patients, there was any association between gMB, uremic toxins, inflammation and oxidative stress. Methods: We analyzed gMB composition, uremic toxins (indoxyl sulphate and p-cresyl sulphate), inflammatory cytokines (interleukin 10, tumor necrosis factor α, interleukin 6, interleukin 17, interleukin 12 p70, monocyte chemoattractant protein-1 and fetuin-A) and oxidative stress (malondialdehyde) of 64 elderly CKD patients (10 &lt, eGFR &lt, 45 mL/min/1.73 m2, not on dialysis) categorized as sarcopenic and not-sarcopenic. Sarcopenia was defined according to European Working Group on Sarcopenia in Older People 2 criteria. Results: Sarcopenic patients had a greater abundance of the Micrococcaceae and Verrucomicrobiaceae families and of Megasphaera, Rothia, Veillonella, Akkermansia and Coprobacillus genera. They had a lower abundance of the Gemellaceae and Veillonellaceae families and of Acidaminococcus and Gemella genera. GMB was associated with uremic toxins, inflammatory cytokines and MDA. However, uremic toxins, inflammatory cytokines and MDA were not different in sarcopenic compared with not-sarcopenic individuals, except for interleukin 10, which was higher in not-sarcopenic patients. Conclusions: In older CKD patients, gMB was different in sarcopenic than in not-sarcopenic ones. Several bacterial families and genera were associated with uremic toxins and inflammatory cytokines, although none of these latter substantially different in sarcopenic versus not-sarcopenic patients.

Background in patients with chronic kidney disease (CKD) indoxyl sulfate (IS) and p-cresyl sulfate (PCs) may induce sarcopenia either directly or via systemic inflammation. We evaluated whether IS and PCs were associated with: sarcopenia, systemic inflammation and nutritional status. Methods: we examined cross sectionally 93 patients with advanced CKD. Sarcopenia was identified according to EWGSOP2 definition. Malnutrition was assessed by Malnutrition Inflammation Score (MIS) and Protein Energy Wasting syndrome (PEW). Inflammatory status was assessed by dosing: CRP, IL6, TNFα, MCP1, IL10, IL17, IL12p70. Results: we did not find any association of sarcopenia with IS and PCs. IS was associated with LogTNFα and LogMCP-1 in the overall cohort (r = 0.30, p = 0.0043; r = 0.22 p = 0.047) and in not sarcopenic patients (r = 0.32, p = 0.0077; r = 0.25, p = 0.041). PCs was associated with LogIL10 and LogIL12p70 in sarcopenic patients (r = 0.58, p = 0.0042; r = 0.52, p = 0.013). IS was higher in patients without PEW (p = 0.029), while PCs was higher in patients with PEW (p = 0.0040). IS and PCs were not different in patients with normal or increased MIS. Conclusions: IS and PCs were not associated with sarcopenia, although they were both associated with some inflammatory pathways. Notably, we found a positive association of PCs with PEW syndrome.

Background: In patients with chronic kidney disease (CKD), sarcopenia can be determined by a wide spectrum of risk factors. We evaluated the association of sarcopenia with nutritional, behavioral and inflammatory patterns in older patients with advanced CKD. Methods: we cross-sectionally evaluated 113 patients with CKD stages 3b-5. Sarcopenia was defined according to the EWGSOP2 criteria. We assessed: anthropometry, bioelectrical impedance analysis, physical, and psychological performance. Nutritional status was assessed using the Malnutrition Inflammation Score (MIS) and by verifying the eventual presence Protein Energy Wasting syndrome (PEW). Systemic inflammation was assessed by dosing: CRP, IL6, TNF&alpha, MCP1, IL10, IL17, fetuin, IL12. Results: 24% of patients were sarcopenic. Sarcopenic individuals had lower creatinine clearance (18 &plusmn, 11 vs. 23 &plusmn, 19 mL/min, p = 0.0087) as well as lower BMI (24.8 &plusmn, 3.0 vs. 28.4 &plusmn, 5.5 Kg/m2, p &lt, 0.0001) and a lower FTI (11.6 &plusmn, 3.9 vs. 14.4 &plusmn, 5.1 kg/m2, p = 0.023). Sarcopenic persons had higher prevalence of PEW (52 vs. 20%, p &lt, 0.0001) and a tendency to have higher MIS (6.6 &plusmn, 6.5 vs. 4.5 &plusmn, 4.0, p = 0.09), however, they did not show any difference in systemic inflammation compared to non-sarcopenic individuals. Conclusions: CKD sarcopenic patients were more malnourished than non-sarcopenic ones, but the two groups did not show any difference in systemic inflammation.

The presence of circulating permeability factors (cPFs) has been hypothesized to be associated with recurrence of focal segmental glomerulosclerosis (rFSGS) in renal allografts. The available methods to detect cPFs are complex, not easily repeatable and inappropriate to represent the anatomical characteristics of the three-layer glomerular filtration barrier (GFB). Here we describe a novel method which measures the permeability to bovine serum albumin (BSA) through a three-layer device (3LD). The 3 layers comprise: (1) conditionally immortalized human podocytes (HCiPodo), (2) collagen type IV coated porous membrane and (3) human glomerular endothelial cells (HCiGEnC). Using this method, we found that sera from all rFSGS patients increased albumin permeability, while sera from non recurrent (nrFSGS) and genetic (gFSGS) forms of FSGS did not. The mechanisms underlying the increase of albumin permeability are probably due to endothelial cell damage as an initial event, which was demonstrated by the decrease of Platelet endothelial cell adhesion molecule (PECAM-1 or CD31), while the podocytes’ expressions of synaptopodin and podocin were normal. Furthermore, we also found that the plasmapheretic treatment (PPT) eliminated the effect of increasing BSA permeability in sera from rFSGS patients. These preliminary data suggest that our in vitro GFB model could not only be useful in predicting the recurrence of FSGS after renal transplantation (RTx), but also be a valuable in vitro model to study podocyte and endothelial cell biology.

Idiopathic focal segmental glomerulosclerosis (FSGS) is a progressive and proteinuric kidney disease that starts with podocyte injury. Podocytes cover the external side of the glomerular capillary by a complex web of primary and secondary ramifications. Similar to dendritic spines of neuronal cells, podocyte processes rely on a dynamic actin-based cytoskeletal architecture to maintain shape and function. Brain-derived neurotrophic factor (BDNF) is a pleiotropic neurotrophin that binds to the tropomyosin-related kinase B receptor (TrkB) and has crucial roles in neuron maturation, survival, and activity. In neuronal cultures, exogenously added BDNF increases the number and size of dendritic spines. In animal models, BDNF administration is beneficial in both central and peripheral nervous system disorders. Here we show that BDNF has a TrkB-dependent trophic activity on podocyte cell processes; by affecting microRNA-134 and microRNA-132 signalling, BDNF up-regulates Limk1 translation and phosphorylation, and increases cofilin phosphorylation, which results in actin polymerization. Importantly, BDNF effectively repairs podocyte damage in vitro, and contrasts proteinuria and glomerular lesions in in vivo models of FSGS, opening a potential new perspective to the treatment of podocyte disorders.

Podocytes are postmitotic renal glomerular cells with multiple ramifications that extend from the cell body. Processes departing from a podocyte interdigitate with corresponding projections from neighboring cells and form an intricate web that enwraps the glomerular capillary completely. Podocyte processes are interconnected by the slit diaphragm, an adhesion junction mostly formed by Ig-like molecules, cadherins/protocadherins, ephrin/eph, and neurexin molecules organized in an assembly that resembles synaptic junctions. Podocyte failure is primarily or secondarily implicated in all forms of proteinuric glomerular diseases, as confirmed by the morphological changes of their elaborate cell architecture detectable by electron microscopy. Importantly, mutations of podocyte proteins are responsible for the most severe forms of congenital nephrotic syndrome. In the last 15 years, progressive technological advances have aided the study of podocyte biology and pathology, confirming the relevance of podocyte molecules and signaling pathways for the function of the glomerular filter. This review will examine the most important and newest discoveries in the field, which is rapidly evolving, hopefully leading to a detailed knowledge of this fascinating cell and to the development of specific therapeutic options for proteinuric diseases.

Refers to: D. Mattinzoli, M.P. Rastaldi, M. Ikehata, S. Armelloni, C. Pignatari, L.A. Giardino, M. Li, C.M. Alfieri, A. Regalia, D. Riccardi, P. Messa FGF23-regulated production of Fetuin-A (AHSG) in osteocytes Bone, Volume 83, February 2016, Pages 35-47

Chronic kidney disease (CKD) is a disabling condition associated with different medical comorbidities including depression and cognitive impairment. We investigated the association between malnutrition, inflammation and depressive/cognitive symptoms in elderly subjects with advanced CKD.We evaluated cross-sectionally 132 elderly subjects (age ≥65 years) with advanced CKD (stage 4-5, non-dialytic-ND) in regular follow up at the outpatient clinic of nephrology. Blood and urinary samples were collected after an overnight fast. All patients were evaluated by Geriatric Depression Scale (GDS)-30 items for severity of depressive symptoms, Mini Mental State Examination (MMSE) and the Clock Drawing Test (CDT) for cognition. Nutritional status was assessed by Malnutrition Inflammation Score (MIS). Different linear regression models were performed to study the association between clinical variables, diet and inflammatory parameters with the above mentioned rating scale scores. A final linear regression model with only previous statistically significant variables was performed for GDS scores.Our cohort consisted of 95 males and 37 females with a mean age of 78 ± 7. Female gender (B = 3.20, p .01), higher MIS (B = 0.29, p = .02) and higher IL-12p70 serum levels (pg/mL) (B = 0.37, p = .03) were associated with severity of depressive symptoms. MIS was associated with the severity of cognitive impairment as assessed by MMSE (B = -0.19, p .01) and CDT (B = 0.10, p = .03).In elderly subjects affected by CKD the severity of depressive symptoms and cognitive impairment is associated with specific inflammatory and nutritional parameters. These results have to be considered as preliminary and need replication by further studies.

Introduction: AHSG, a serum glycoprotein with recognized anti-calcification activity, has also been suggested to modulate both bone formation and resorption. Though the bulk of AHSG is mostly synthesized in the liver, it has been claimed that also bone cells might produce it. However, the extent of the bone AHSG production and the potential controlling factors remain to be definitively proven.\ud \ud A relevant number of studies support the notion that FGF23, a bone-derived hormone, not only regulates the most important mineral metabolism (MM) related factors (phosphate, parathyroid hormone, vitamin D, etc.), but might be also involved in cardiovascular (CV) outcome, both in chronic kidney disease (CKD) patients and in the general population. Furthermore, in addition to some direct autocrine and paracrine effects in bone, FGF23 has been suggested to interact with AHSG.\ud \ud In this study we investigated if AHSG is really produced by bone cells, and if its bone production is related and/or controlled by FGF23, using cultured bone cells, according to a new method recently published by our group.\ud \ud Results: Our data show that AHSG is consistently produced in osteocytes and to a far lesser extent in osteoblasts. Both FGF23 addition to the culture medium and its over-expression in osteocytes were associated with a consistent increase of both AHSG mRNA and protein, while FGF23 silencing was followed by opposite effects. Though most of these results were largely affected by the blockage of FGF23 receptors, the role of these receptors in the different experimental sets is still not completely clarified. In addition, we found that FGF23 and AHSG proteins co-localized both in cytoplasm and nucleus, which suggests a possible reciprocal interactivity.\ud \ud Conclusions: Our data not only confirm that AHSG is produced in bone, mainly in osteocytes, but show for the first time that its production is modulated by FGF23. Since both proteins play important roles in the bone and cardiovascular pathology, these results add new pieces to the puzzling relationship between bone and vascular pathology, in particular in CKD patients, prompting future investigations in this field.

Proteinuria is a common symptom of glomerular diseases and is due to leakage of proteins from the glomerular filtration barrier, a three-layer structure composed by two post-mitotic highly specialized and interdependent cell populations, i.e. glomerular endothelial cells and podocytes, and the basement membrane in between. Despite enormous progresses made in the last years, pathogenesis of proteinuria remains to be completely uncovered. Studies in the field could largely benefit from an in vitro model of the glomerular filter, but such a system has proved difficult to realize. Here we describe a method to obtain and utilize a three-dimensional podocyte-endothelial co-culture which can be largely adopted by the scientific community because it does not rely on special instruments nor on the synthesis of devoted biomaterials. The device is composed by a porous membrane coated on both sides with type IV collagen. Adhesion of podocytes on the upper side of the membrane has to be preceded by VEGF-induced maturation of endothelial cells on the lower side. The co-culture can be assembled with podocyte cell lines as well as with primary podocytes, extending the use to cells derived from transgenic mice. An albumin permeability assay has been extensively validated and applied as functional readout, enabling rapid drug testing. Additionally, the bottom of the well can be populated with a third cell type, which multiplies the possibilities of analyzing more complex glomerular intercellular signaling events. In conclusion, the ease of assembly and versatility of use are the major advantages of this three-dimensional model of the glomerular filtration barrier over existing methods. The possibility to run a functional test that reliably measures albumin permeability makes the device a valid companion in several research applications ranging from drug screening to intercellular signaling studies.

Progresses in podocyte biology have been strictly connected with genetic advances; the identification of genes mutated in familial and sporadic forms of nephrotic syndrome has been followed by functional studies of the encoded proteins, revealing numerous properties of the cell. The molecules uncovered so far belong to three main categories: a) proteins located at the slit diaphragm, the intercellular junction which laterally connects podocyte processes and is responsible for selectivity of the glomerular filter, b) molecules involved in regulation of actin dynamics, which are essential for the maintenance of podocyte structure and function, and c) molecules belonging to intracellular organelles, such as mitochondria and lysosomes, which are central players in podocyte metabolism. Considering the key role of the podocyte in health and disease of the glomerular filter, better knowledge of this cell is a pre-requisite for developing targeted therapies of glomerular diseases.

Nephrin is an immunoglobulin-like adhesion molecule first discovered as a major component of the podocyte slit diaphragm, where its integrity is essential to the function of the glomerular filtration barrier. Outside the kidney, nephrin has been shown in other restricted locations, most notably in the central nervous system (CNS) of embryonic and newborn rodents. With the aim of better characterizing nephrin expression and its role in the CNS of adult rodents, we studied its expression pattern and possible binding partners in CNS tissues and cultured neuronal cells and compared these data to those obtained in control renal tissues and podocyte cell cultures. Our results show that, besides a number of locations already found in embryos and newborns, endogenous nephrin in adult rodent CNS extends to the pons and corpus callosum and is expressed by granule cells and Purkinje cells of the cerebellum, with a characteristic alternating expression pattern. In primary neuronal cells we find nephrin expression close to synaptic proteins and demonstrate that nephrin co-immunoprecipitates with Fyn kinase, glutamate receptors and the scaffolding molecule PSD95, an assembly that is reminiscent of those made by synaptic adhesion molecules. This role seems to be confirmed by our findings of impaired maturation and reduced glutamate exocytosis occurring in Neuro2A cells upon nephrin silencing. Of note, we disclose that the very same nephrin interactions occur in renal glomeruli and cultured podocytes, supporting our hypothesis that podocytes organize and use similar molecular intercellular signalling modules to those used by neuronal cells.

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line. The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features. After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules. Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models. The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.

Injection of DEAE dextran into Lewis rats can produce proteinuria and has been reported as a model of IgA nephropathy.Cationic diethyl aminoethyl (DEAE) dextran of molecular weight 500 kDa was injected into male Lewis rats. After a pre-immunization period of 3 weeks, the animals were divided into two groups: group 1 (n = 14) received daily i.v, injections of 3.5 mg of antigen, group 2 (n = 14) was injected with 1.5 mg three times per week for a total period of 6 weeks. I.v. treatment was initiated with gradually increasing doses of DEAE dextran in both groups for 1 week, after which the maintenance dose was reached.We observed the appearance of proteinuria in a nephrotic range after 5 weeks of i.v. injections in group 1 (urinary excretion: 332 +/- 83 mg/24 h, controls: 53 +/- 14 mg/24 h). In group 2, the proteinuria was almost equal to protein excretion of healthy rats of the same weight (67 +/- 20 mg/24 h). The serum and urine creatinine were normal. By light microscopy of kidney biopsies, the presence of focal and segmental proliferation of mesangial cells after 6 weeks of i.v. injections was identified. Immunohistochemistry revealed no deposition of IgA, IgM, IgG, or C3. Using anti-ED1 antibodies, there was no evidence of interstitial infiltration of monocytes/macrophages after 6 weeks of i.v. injections. Staining for proliferating cell nuclear antigen (PCNA) did not show the presence of proliferating cells either in glomeruli or in the interstitium. Staining with FITC-WGA lectin revealed focal and segmental loss of the negative charge in the capillary wall. By electron microscopy there was deposition of dextran in the basal membrane and segmental and focal damage of the podocyte foot processes. As the chemokine RANTES may be involved in glomerular injury, we examined the kidneys of proteinuric and non-proteinuric rats for the presence of RANTES. By indirect immunofluorescence only the proteinuric rats showed RANTES deposition in the mesangium.Injection of rats with DEAE dextran leads to dose-dependent proteinuria without deposition of immune complexes but with podocyte damage. This is associated with local expression of the chemokine RANTES which may play a role in proteinuria of glomerular disease.

In the renal glomerulus, podocytes envelop the external side of the capillary basement membrane with their intertwining ramifications, and ensure elimination of metabolic waste within the urine, while proteins and important blood components are retained into the circulation. To preserve the integrity of the glomerular filter, which is constantly exposed to a high variety of stimuli, podocytes need to communicate by rapid and precise signaling, likely similar to that used by neuronal cells. In the last years, we and others have shown that podocytes are indeed molecularly equipped for communicating in a synaptic-like way, where glutamate and its receptors seem to have a pivotal role, because altering glutamatergic communication leads to podocyte damage and increased filter permeability. Major components of glutamatergic signaling are organized at foot process junctions by adhesion molecules, chiefly by nephrin, and are connected to the actin cytoskeleton, that governs the health of podocytes. Further advances in understanding podocyte physiological behavior and signaling properties have the potential to improve the knowledge of podocyte diseases, first among them idiopathic focal segmental glomerulosclerosis that still needs more precise molecular-based diagnosis and targeted treatment.

Background/Aims: The relative contribution of genetic factors and dietary patterns to glomerular damage in healthy individuals and prediabetic conditions is currently unclear. All Rab3A knockout (KO) mice spontaneously develop macroalbuminuria, but only male mice exhibit a glucose-intolerant phenotype, thus making the model suitable to examine the impact of a diet on preexisting podocyte damage. Methods: Male and female Rab3A KO and wild-type (WT) mice were chronically fed a high-glucose diet (HGD). Biochemical tests, histology and immunohistochemistry were periodically performed whilst primary podocytes served for in vitro analyses. Results: Chronic administration of an HGD did not induce de novo alterations in WT kidneys but caused progressive worsening of podocyte and glomerular damage in both male and female Rab3A KO. Though glomerular lesions, reminiscent of human diabetic nephropathy, were more severe in male mice, overt proteinuria and renal damage were also evident in female mice. The in vitro analysis of Rab3A WT and KO podocytes revealed diminished actin plasticity in the cell processes of KO podocytes. Furthermore, a modest increase in glucose concentration induced profound cytoskeletal changes only in Rab3A KO cells. Conclusions: Our data show that chronic administration of an HGD to Rab3A KO mice that have a genetic defect that impairs podocyte actin plasticity results in increased podocyte damage and leads to overt proteinuria. If the same diet is given to male Rab3A KO animals, with additionally altered glucose homeostasis, this results in renal lesions similar to those of human diabetic nephropathy.

Adducins are cytoskeletal actin-binding proteins (α, β, γ) that function as heterodimers and heterotetramers and are encoded by distinct genes. Experimental and clinical evidence implicates α- and β-adducin variants in hypertension and renal dysfunction. Here, we have addressed the role of α- and β-adducin on glomerular function and disease using β-adducin null mice, congenic substrains for α- and β-adducin from the Milan hypertensive (MHS) and Milan normotensive (MNS) rats and patients with IgA nephropathy. Targeted deletion of β-adducin in mice reduced urinary protein excretion, preceded by an increase of podocyte protein expression (phospho-nephrin, synaptopodin, α-actinin, ZO-1, Fyn). The introgression of polymorphic MHS β-adducin locus into MNS (Add2, 529R) rats was associated with an early reduction of podocyte protein expression (nephrin, synaptopodin, α-actinin, ZO-1, podocin, Fyn), followed by severe glomerular and interstitial lesions and increased urinary protein excretion. These alterations were markedly attenuated when the polymorphic MHS α-adducin locus was also present (Add1, 316Y). In patients with IgA nephropathy, the rate of decline of renal function over time was associated to polymorphic β-adducin (ADD2, 1797T, rs4984) with a significant interaction with α-adducin (ADD1, 460W, rs4961). These findings suggest that adducin genetic variants participate in the development of glomerular lesions by modulating the expression of specific podocyte proteins. Electronic supplementary material The online version of this article (doi:10.1007/s00109-009-0549-x) contains supplementary material, which is available to authorized users.

The metabotropic glutamate (mGlu) receptor 1 (GRM1) has been shown to play an important role in neuronal cells by triggering, through calcium release from intracellular stores, various signaling pathways that finally modulate neuron excitability, synaptic plasticity, and mechanisms of feedback regulation of neurotransmitter release. Herein, we show that Grm1 is expressed in glomerular podocytes and that a glomerular phenotype is exhibited by Grm1(crv4) mice carrying a spontaneous recessive inactivating mutation of the gene. Homozygous Grm1(crv4/crv4) and, to a lesser extent, heterozygous mice show albuminuria, podocyte foot process effacement, and reduced levels of nephrin and other proteins known to contribute to the maintenance of podocyte cell structure. Overall, the present data extend the role of mGlu1 receptor to the glomerular filtration barrier. The regulatory action of mGlu1 receptor in dendritic spine morphology and in the control of glutamate release is well acknowledged in neuronal cells. Analogously, we speculate that mGlu1 receptor may regulate foot process morphology and intercellular signaling in the podocyte.

Podocytes possess the complete machinery for glutamatergic signaling, raising the possibility that neuron-like signaling contributes to glomerular function. To test this, we studied mice and cells lacking Rab3A, a small GTPase that regulates glutamate exocytosis. In addition, we blocked the glutamate ionotropic N-methyl-d-aspartate receptor (NMDAR) with specific antagonists. In mice, the absence of Rab3A and blockade of NMDAR both associated with an increased urinary albumin/creatinine ratio. In humans, NMDAR blockade, obtained by addition of ketamine to general anesthesia, also had an albuminuric effect. In vitro, Rab3A-null podocytes displayed a dysregulated release of glutamate with higher rates of spontaneous exocytosis, explained by a reduction in Rab3A effectors resulting in freedom of vesicles from the actin cytoskeleton. In addition, NMDAR antagonism led to profound cytoskeletal remodeling and redistribution of nephrin in cultured podocytes; the addition of the agonist NMDA reversed these changes. In summary, these results suggest that glutamatergic signaling driven by podocytes contributes to the integrity of the glomerular filtration barrier and that derangements in this signaling may lead to proteinuric renal diseases.

To determine the immunosuppressive effect(s) of cephalosporin cefonicid (CEFO) on human T-helper cells (Th), we exposed human peripheral blood mononuclear cells (PBMC) to various concentrations of CEFO during in vitro stimulation with a panel of T-lymphocyte stimulators that activate different Th/antigen presenting cell (APC) pathways. We evaluated the proliferation and IL-2 production induced by influenza virus (FLU), allogeneic lymphocytes (ALLO), xenogeneic mouse splenocytes (XENO) or phytohemagglutinin (PHA). The proliferative responses to FLU and XENO were much more depressed by CEFO than those to ALLO or PHA. After 7 days of culture with the highest dose of CEFO tested (200 mg/l) the stimulation index (stimulated/unstimulated culture) was near to 0 in FLU and XENO treated cultures, indicating that the response against these antigens was completely abrogated. The responses to ALLO and PHA were also impaired, but not abrogated (stimulation index greater than 1). Since FLU and XENO utilize the CD4+ Th/self-APC pathway our data suggested that this pathway was extremely sensitive to CEFO-induced inhibition both when the response requires memory Th cells (FLU) and virgin Th cells (XENO). The incubation with CEFO (200 mg/l) reduced the IL-2 production by XENO, FLU and ALLO to less than 20% of control cultures, while paradoxically increases to 120% the production by PHA.

Diabetic nephropathy (DN) is one of the major complications of diabetes and the most common cause of ESRD. Cell death by apoptosis might contribute to the gradual loss of renal mass in DN. In fact, apoptosis has been detected in tubular epithelial, endothelial, and interstitial cells of renal biopsy samples from patients with DN.1,2 Cell death via apoptosis is an active response of cells to altered microenvironments and is characterized by the activation of specific intracellular pathways. Hyperglycemia is among the microenvironmental factors that may induce or facilitate apoptosis. A high glucose concentration, per se, promotes apoptosis in a variety of cell types, including renal tubular epithelium.3–5 Lethal cytokines from the TNF superfamily activate death receptors on the cell surface with subsequent activation of caspases, central activators, and effectors of apoptosis.6 The apoptotic process is modulated by a host of checks and balances with a multitude of positive and negative regulators.6 To display the apoptosis network that is active in human DN in a comprehensive manner, we have studied the molecular mechanisms underlying tubulointerstitial apoptosis in the course of DN by monitoring changes in the renal transcriptome of the diabetic kidney. The combination of unbiased gene expression profiling with focused data mining has proved to be a powerful tool to expand our knowledge of relevant pathways and players in human disease.7 Applying gene ontology category “cell death” followed by pathway mapping to the genome-wide data sets defined OPG and TRAIL as key nodes regulated in the transcriptomic profile. TNF-related apoptosis–inducing ligand (TRAIL/APO2L/TNFSF10)8 is an atypical member of the TNF gene superfamily because it is capable of binding a complex system of receptors. Two of them, TRAIL-R1 (DR4/TNFRSF10A) and TRAIL-R2 (DR5/TRICK2/KILLER/TNFSFR10B), contain a cytoplasmic death domain and transmit an apoptotic signal in response to TRAIL.9 Two others, TRAIL-R3 (TRID/DcR1/TNFRSF10C) and TRAIL-R4 (DcR2/TNFRSF10D), bind TRAIL without activation of the apoptotic machinery and seem to antagonize the death domain–containing TRAIL receptors.10 In addition, osteoprotegerin (OPG/TNFRSF11B), a regulator of osteoclastogenesis that binds to the TNF superfamily member RANKL,11 is a soluble decoy receptor for TRAIL.12 We have found that TRAIL expression is altered in the DN kidney. Tubulointerstitial TRAIL expression is increased in both mRNA and protein in DN samples. The functional relevance of these changes in TRAIL expression has been studied using cultured renal tubular cells that express all relevant elements of the TRAIL pathway. Because we identified a functionally significant activation of the NF-κB pathway in DN in a parallel study,7 we evaluated the interplay of the TRAIL pathway, NF-κB, and cell death in tubular cells. TRAIL was found to activate NF-κB, and inhibition of NF-κB activation sensitized cells to TRAIL-induced apoptosis.

Rats of the Milan hypertensive strain (MHS) are resistant to both hypertensive and diabetic renal disease. Genetically determined hypertrophy of intrarenal arteries has been suggested as the putative mechanism preventing transmission of systemic hypertension to the glomerular microcirculation or diabetes-induced loss of autoregulation, which lead to glomerular hypertension and consequent podocyte injury and proteinuria. This study aimed to investigate glomerular barrier function and structure in ageing and diabetic MHS rats under basal conditions and after injection of 2.5 g of bovine serum albumin (BSA) causing increased workload and possibly removing haemodynamic protection by inducing renal cortical vasodilatation. Genetically related rats of the Milan normotensive strain (MNS) served as a proteinuric counterpart. No change in renal function or structure was detected in diabetic MHS rats, whereas MNS rats developed diabetic nephropathy superimposed on that occurring spontaneously in this strain. Diabetic, but not non-diabetic, MHS rats showed significantly reduced synaptopodin and nephrin expression, though to a lesser extent than non-diabetic and diabetic MNS rats, together with unchanged podocyte number, density and structure and no proteinuria. Agrin expression was significantly altered in diabetic versus non-diabetic MHS animals, whereas collagen I was expressed only in diabetic MHS rats and collagen IV content did not change significantly between the two groups. Upon BSA injection, proteinuria increased markedly and abundant BSA was detected only in kidneys from diabetic MHS rats. BSA injection was associated with changes in intrarenal arteries suggesting vasodilatation, without any influx of inflammatory cells. These data indicate that while MNS rats show marked changes in the glomerular filtration barrier with either age or diabetes, glomerulosclerosis-resistant MHS rats develop only minor diabetes-induced podocyte (and extracellular matrix) alterations, which are not associated with proteinuria unless they are unmasked by an increased workload or removal of the haemodynamic protection.

Although patients with chronic renal failure are increasing worldwide, many aspects of kidney biology remain to be elucidated. Recent research has uncovered several molecular properties of the glomerular filtration barrier, in which podocytes, highly differentiated, ramified cells that enwrap the glomerular basement membrane, have been reported to be mainly responsible for filter's selectivity. We previously described that podocytes express Rab3A, a GTPase restricted to cell types that are capable of highly regulated exocytosis, such as neuronal cells. Here, we first demonstrate by a proteomic study that Rab3A in podocytes coimmmunoprecipitates with molecules once thought to be synapse specific. We then show that podocytes possess structures resembling synaptic vesicles, which contain glutamate, coexpress Rab3A and synaptotagmin 1, and undergo spontaneous and stimulated exocytosis and recycling, with glutamate release. Finally, from the results of a cDNA microarray study, we describe the presence of a series of neuron- and synapse-specific molecules in normal human glomeruli and confirm the glomerular protein expression of both metabotropic and ionotropic glutamate receptors. These data point toward a synaptic-like mechanism of communication among glomerular cells, which perfectly fits with the molecular composition of the glomerular filter and puts in perspective several previous observations, proposing a different working hypothesis for understanding glomerular signaling dynamics.

Membranous glomerulonephritis (MGN), histologically defined by subepithelial immune deposits, is the most common cause of nephrotic syndrome in Caucasian adults. The current hypothesis of the underlying disease mechanism postulates production of antibodies against podocyte-derived antigens. Respective antigens could be demonstrated in different animal models and recently in human neonatal MGN. Further support for this hypothesis was generated by the response of human MGN to therapeutic B cell depletion by rituximab. However, the role of B cells in this disease is not well defined. In this study, the interstitial expression of CD20 mRNA was determined in 31 MGN patients and controls (tumor nephrectomies (n = 4), minimal change disease (MCD, n = 10) and focal segmental glomerulosclerosis (n = 6)). CD20 mRNA expression was significantly higher in MGN patients compared to controls. By immunohistochemistry, a focal or diffuse interstitial B cell infiltration could be detected in MGN patients (n = 63), which was absent or minimal in patients with MCD (n = 11). These data suggest an involvement of B cells in the pathogenesis of MGN, possibly as antigen-presenting cells. Further studies should investigate the potential to predict the response to therapeutic B cell depletion by intrarenal CD20 quantification, a potential diagnostic basis for the selection of a specific therapy currently evolving for renal disease.

The zebrafish pronephros is gaining popularity in the nephrology community, because embryos are easy to cultivate in multiwell plates, allowing large number of experiments to be conducted in an in vivo model. In a few days, glomeruli reach complete development, with a structure that is similar to that of the mammalian counterpart, showing a fenestrated endothelium and a basement membrane covered by the multiple ramifications of mature podocytes. As a further advantage, zebrafish embryos are permeable to low molecular compounds, and this explains their extensive use in drug efficacy and toxicity experiments. Here we show that low concentrations of adriamycin (i.e. 10 and 20 µM), when dissolved in the medium of zebrafish embryos at 9 hours post-fertilization and removed after 48 hours (57 hpf), alter the development of podocytes with subsequent functional impairment, demonstrated by onset of pericardial edema and reduction of expression of the podocyte proteins nephrin and wt1. Podocyte damage is morphologically confirmed by electron microscopy and functionally supported by increased clearance of microinjected 70 kDa fluorescent dextran. Importantly, besides pericardial edema and glomerular damage, which persist and worsen after adriamycin removal from the medium, larvae exposed to adriamycin 10 and 20 µM do not show any myocardiocyte alterations nor vascular changes. The only extra-renal effect is a transient delay of cartilage formation that rapidly recovers once adriamycin is removed. In summary, this low dose adriamycin model can be applied to analyze podocyte developmental defects, such as those observed in congenital nephrotic syndrome, and can be taken in consideration for pharmacological studies of severe early podocyte injury.

Cryoglobulinemic glomerulonephritis is particularly frequent in type II mixed IgMk-IgG cryoglobulinemia. The typical form is a membranoproliferative glomerulonephritis with a particular monocyte infiltration. In the most severe cases, there is occlusion of the capillary lumina by the same immunoglobulin constituents of the cryoprecipitate. While it is generally accepted that the “hyaline thrombi” are endoluminal aggregates of IgG-IgM immune complexes, probably favored by high endocapillary concentration of cryoglobulins, the modality of generation has not been studied. To study the dynamic formation of such “thrombi,” we reproduced an experimental model of cryoglobulinemic glomerulonephritis in mice by injecting them twice a day for 3 days with 4 mg human IgMk-IgG cryoglobulins previously solubilized at 37 degrees C. The dynamic formation of immunodeposits was studied by immunofluorescence and electron microscopy. After 1 day, only mesangial deposits were found; after 3 days, in addition to mesangial deposition, all the capillary lumina were occluded by huge electron-dense bodies. To look for and quantify the contacts between such “thrombi” and mesangial or subendothelial deposits, we obtained serial, ultrathin, 0.5-microm sections that allowed us to reconstruct the whole glomerular tuft. Within each serial section, there was continuity between hyaline thrombi and mesangial or subendothelial deposits ranging from 80% to 85% of the capillary loops. The percentage was 100% for two adjacent serial sections. In conclusion, our data demonstrate directly for the first time that hyaline thrombi follow mesangial deposits. The high percentage of contacts between thrombi and mesangial or subendothelial deposits suggests that they result from in situ build-up of true huge endoluminal immunodeposits after saturation of the clearance capacity of the mesangium. (Am J Kidney Dis 1998 Mar;31(3):435-42)

In our previous experimental work we suggested that the frequent nephritogenicity of type II cryoglobulins could depend on a particular affinity of the immunoglobulin (Ig) M kappa rheumatoid factor (RF) component for mesangial matrix. Since cellular fibronectin (cFN) in the human kidney is mainly represented in glomerular mesangium, we studied the binding capacity to cFN of IgM kappa RFs from type II cryoglobulins compared with other different monoclonal and polyclonal IgM and IgM RFs. We purified 13 IGM kappa from human IgM kappa/IgG cryoglobulins, eight monoclonal IgM from patients with Waldenström's macroglobulinemia, nine polyclonal IgM from normal donors, and eight polyclonal IgM RFs from patients with rheumatoid arthritis. Purified IgM were used at the same concentration in enzyme-linked immunosorbent assay (ELISA) on cFN-coated plates. All the cryoglobulin IgM showed high specific binding to cFN while IgM from Waldenström's macroglobulinemia, normal IgM, and polyclonal IgM RFs had low or absent binding. These data were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cFN followed by Western blot analysis with purified IgM. The IgM kappa binding to cFN persisted using IgM kappa monomers, and was inhibited by cFN but not by plasma FN in a specific inhibition test. Further enzyme-linked immunosorbent assay studies showed that cryoglobulin IgM kappa RFs are still able to bind IgG in a dose-dependent manner once linked to solid-phase cFN. The data suggest that the affinity of cryoglobulin IgM kappa RFs for immobilized cFN could be involved in the particular high nephritogenicity of type II cryoglobulins and might lead to in situ immune complex formation.

BACKGROUND: Human cryoglobulinemia is sometimes associated with glomerulonephritis (GN) due to deposition of cryoglobulins (cryos). To see whether human cryos can induce GN in mice and to study time-related changes of glomerular lesions and possible factors of cryos' nephritogenicity, we developed an experimental passive model of cryoglobulinemic GN. EXPERIMENTAL DESIGN: Two cryos IgMk-IgG from 2 patients with active GN (OLD and SOR), 2 cryos IgMk-IgG (TAC and GRO) and 1 IgMλ (CHI) from 3 patients without GN were purified, solubilized at 37° C and injected intravenously into BALB/c mice, 4 mg, twice a day. To study the possible factors of cryo nephritogenicity, we analyzed: (a) the presence, amount, and size of complexed IgMk-IgG at 37° C by fast flow liquid chromatography; (b) the Cc1 or Lc1 subclass of rheumatoid factors; (c) the isoelectric points of the IgMks; (d) The proportion of IgG subclasses in cryos. RESULTS: On day 1 from the beginning of intravenous injections, cryos OLD had induced mesangial deposits of human IgM, human IgG, mouse C3 and mesangial hypercellularity. On day 2, phagocytizing cells were found along with massive endoluminal and subendothelial deposits of IgM, IgG, and C3. On day 6, perivascular infiltrates of mononuclear cells were also seen. Cryos SOR induced a similar but milder form of GN. After administration of purified OLD IgMk, OLD IgG, GRO IgMk or GRO IgG, only OLD IgMk was deposited in the mesangium. Analysis of all the cryos revealed that: the amount of complexed IgMk-IgG at 37° C was always less than 1% of cryos; Cc1 and Lc1 idiotypes were not related to the nephritogenicity of cryos, the isoelectric points of IgMks were 4.5 to 5.5 and IgG1 was the prevalent subclass. CONCLUSIONS: Data demonstrate that human cryos from patients with GN can induce GN in mice that resembles the corresponding human pathology. The affinity of IgMk for glomeruli and the unexpectedly small amounts of IgMk-IgG complexes at 37° C suggest that there is a role of in situ binding in nephritogenicity which is independent of the isoelectric point, rheumatoid factor idiotype, or IgG subclass