Glomerular Protein Research Articles

Protein energy wasting and long-term outcomes in nondialysis dependent chronic kidney disease.

Nutritional status and protein energy wasting (PEW) is prevalent in patients with nondialysis-dependent chronic kidney disease (CKD). The relationship between PEW and long-term development of clinically important outcomes remains to be examined. To investigate the relationships between PEW, as measured by Subjective Global Assessment (SGA 1-7), and progression to important clinical outcomes: mortality and/or kidney failure. Prospective cohort design. One hundred and thirty-nine participants werewell-nourished and 37 moderately malnourished patients with CKD 3-5. The outcomes were 2, 5, and 10-year progression to kidney failure (dialysis or transplant) or mortality, kidney failure alone, and mortality alone. SGA was determined by a registered renal dietitian. Food frequency questionnaires were used to assess dietary intake. Clinical and laboratory baseline characteristics were collected. Multivariable regression models and Cox models were created to examine the relationship between SGA and outcomes. PEW was associated with the combined outcome of kidney failure or mortality at 2 (p = 0.003), 5 (p = 0.004), but not at 10 (p = 0.73) years. This relationship was primarily driven by the relationship between PEW and kidney failure. In Cox models, the relationship between PEW and kidney failure remained after adjusting for Kidney Failure Risk Equation scores. The multivariable modeling revealed that PEW remained a statistically significant predictor of the combined outcome and ESKD after adjustment for age, estimated glomerular filtration rate (eGFR), sex, albumin-to-creatinine ratio, diabetes, albumin, and protein intake. PEW, determined by the SGA 1-7, is an important prognostic tool. Further research looking at clinically important outcomes are needed to implement nutritional interventions for nondialysis-dependent CKD patients.

MO610NUTRITIONAL PARAMETERS OF PATIENTS WITH END-STAGE RENAL DISEASE IN THE GENERAL UNIVERSITY HOSPITAL AREA

Abstract Background Malnutrition is one of the problems that patients can develop during the later stages of kidney disease. These problems are derived from uraemia and acidosis that can present due to the decrease in glomerular filtration and added to an accumulation of urine levels. During the months of January 2019 and May 2020 were collected information of 564 patients about age, weight, height, BWI, glomerular filtration rate, use of oral nutrition supplements and albumin, protein, phosphorous and potassium biochemical levels. All of patients were had a GFR below 45 mL/min/1,73m2 and any of them have been started a renal replacement therapy when the data was collected. A descriptive and frequency analysis has been carried out with all the parameters obtained. The T of Student for a related sample test was used due to the normality of the data and the participants condition of intervention – control. Results The results indicate that the mean values for ESRD patients in our area are: age (75,3±12,5 years old), weight (74,7±15,6 kg), height (1,6±0,1 m), IMC (28,8±5,4 kg/m2), glomerular filtration rate (19,9±9,1 mL/min/1,73m2), albumin (3,7±0.5 g/dL), protein (6,8±0,7 g/dL), phosphorous (4,2±1,1 mg/dL) and potassium (4,6±0,5 mEq/L). 41 patients (7,3%) used oral nutrition supplements. We found statistical significance between the stages 4 and 5 of kidney disease and albumin (p-value 0.03 and 0.04) and potassium (p-value 0.04 and 0.04) levels. Only at stage 4 we found statistical significance between the levels of protein (p-value 0.04) and phosphorous (p-value 0.04). Also were found increased levels of albumin (3,73 vs 3,7 mg/dL), proteins (6,87 vs 6,8 mg/dL), phosphorous (4,4 vs 4,3 mg/dL) and potassium (4,8 vs 4,6 mg/dL) in the group who use oral nutrition supplements, however, we did not reach statistical significance between groups. About correlations, we found that the albumin levels have a statistical significance with age, protein, phosphorous and potassium levels, but not with BWI. Conclusion In the General University Hospital area, the patients in ESRD have a high BWI, which can impair the renal disease. Also, we found that BWI decrease in end stages of ESRD worsening the nutritional status before start the renal replacement therapy. About albumin, the worst levels were found at stage 4 of the kidney disease and older patients have more risk of present low albumin levels. The mean values of albumin show that most of our patients meet one of three conditions to diagnose PEW. The protein level was lower at the 5 stage of the disease, and as the albumin, the older patients show lower values of this parameter. Concerning phosphorous and potassium the patients at the end stage of renal disease present higher values of both of them due possibly to the impaired renal function. About the use of nutritional supplements, a low percentage of patients used this as nutritional therapy, but mean levels of biochemical parameters such as albumin, protein, phosphorous and potassium were higher than the patients who did not use them. However, we did not find statistical significance between the use of ONS and better status of biochemical parameters. We can say that older patients in end stage of renal disease have a higher risk of malnutrition in comparison with young patients due to a decrease in BWI, albumin and protein levels. To conclude, the nutritional treatment to these patients should consist, on the one hand, the decrease of the BWI to prevent a quickly loss of renal function and, on the other hand, prevent the malnutrition, especially in the older patients with lower BWI.

FC 019PROTEOMIC PROFILING OF GLOMERULI FROM KIDNEYS WITH HYPERTENSIVE NEPHROPATHY REVEALS SIGNATURE OF DISEASE PROGRESSION

Abstract Background and Aims Hypertensive nephropathy (HN) often represents an unspecific clinical diagnosis applied to non-diabetic, chronic kidney disease (CKD) patients with low-level proteinuria and elevated blood pressure. Kidney biopsy-based findings are the diagnostic gold standard, but they are not entirely specific and there is a need for prognostic markers. We aimed at defining candidate markers that predict disease progression based on protein signatures. Method We included adult patients (n=17) with an eGFR >30 ml/min/1.73m2 and proteinuria <3g/d from the Norwegian Kidney Biopsy Registry (NKBR). Subjects were divided into two groups: stable patients (n=9) and subjects with HN progression (n=8) leading to end-stage renal disease (ESRD) within 20 years of follow-up. Glomerular cross-sections were microdissected from 10 μm whole archival kidney biopsy sections and processed for protein extraction. Proteomic analyses were performed at our local facility PROBE in Bergen using a Q-exactive HF mass spectrometer. Abundances of glomerular proteins were compared between the two groups. Results Amongst a total of 1870 quality filtered proteins, we identified 58 proteins with absolute fold change (FC) ≥1.5, p≤0.05, including 17 proteins with absolute FC ≥2, indicative of HN progression (highest FC: Cadherin 16 and UDP-glucuronosyl-transferase 2B7). Hierarchical cluster and principal component analysis (PCA) with the 17 proteins showed clear separation of samples into these two disease clusters of HN progressors and non-progressors. To find proteins as biomarkers for the identification of progressors, we employed unsupervised K nearest neighbors validation algorithm coupled with leave-one-out internal cross-validation. Thereby, a set of five proteins (incl. cadherin 16) performed best and separated the two groups in a PCA, as shown in Figure 1. This classifier classified 16 of 17 samples correctly (AUC 0.993), misclassifying only one progressor sample. Applying Geneset Enrichment Analysis (GSEA; The Broad Institute, USA), in general metabolic pathways were up-regulated in progressors and structural cell pathways up-regulated in non-progressors. Ingenuity Pathway Analysis (IPA; Qiagen, USA) identified Epithelial Adherens Junction Signaling as the most affected canonical pathway (p=1.95E-06); five of six member proteins were down-regulated in progressors. Conclusion Glomerular proteomic profiling can be used to distinct progressors from non-progressors in HN.

MO087PLASMALEMMAL VESICLE-ASSOCIATED PROTEIN-1 (PLVAP) INDICATES THE FORMATION OF DIAPHRAGM-BRIDGED FENESTRATIONS OF GLOMERULAR ENDOTHELIAL CELLS IN KIDNEY DISEASE

Abstract Background and Aims Plasmalemmal vesicle-associated protein-1 (PLVAP or PV-1) is a major protein of diaphragm-bridged fenestrated endothelial cells found in capillaries of neuroendocrine glands and peritubular capillaries. In contrast to peritubular capillaries, the glomerulus is known for its unique fenestrated endothelium without any diaphragm formation thereby ensuring free filtration. Here we aimed to investigate whether PLVAP is expressed in glomerular endothelial cells in various glomerular diseases and whether PLVAP expression is associated with the formation of diaphragm-bridged endothelial cells. Method A total number of 114 biopsy samples of glomerular diseases including diabetic nephropathy, FSGS, IgA-Nephritis, ANCA-GN and Lupus–Nephritis were analyzed immunohistochemistically for glomerular PLVAP expression. A fraction of PLVAP positive cases was subsequently investigated ultrastrucurally for the formation of diaphragm-bridged glomerular endothelial cells. Results One third of all cases showed at least one glomerulus with one single circumferential PLVAP staining. Interestingly, the most prominent staining, affecting the entire glomerular tuft, was observed in diabetic nephropathy and ANCA-GN. Ultrastructurally, such cases exhibited injured endothelium with focal detachment from the glomerular basement membrane, loss of pore formation and frequently diaphragm-bridged fenestrations reminiscent of peritubular capillaries. Conclusion Our data show that injured glomerular endothelium is capable of forming true diaphragm-bridged fenestrations, suggesting a possible role in preventing glomerular protein leakage and limiting its detachment from the GBM.

MO267HIGHER FRACTIONAL EXCRETION OF TOTAL PROTEIN IS A PREDICTOR OF PROGRESSION IN IDIOPATHIC MEMBRANOUS NEPHROPATHY

Background and AimsLower estimated glomerular filtration rate (eGFR) and higher proteinuria are the most sensitive predictor of the development of progressive renal insufficiency in various glomerular diseases. On the other hand, the onset of idiopathic membranous nephropathy (iMN) shows insidious progression, and the prognosis varies significantly. Therefore, it is difficult to predict the renal outcome in MN, using only the severity of proteinuria. Fractional excretion of total protein (FETP), which was protein clearance divided by creatinine clearance (Ccr), may be a better indicator of protein leak per functioning nephron. A recent study has also reported that FETP accurately predicted transplant failure and was more sensitive and specific than protein creatine ratio (PCR). Few studies, however, have analyzed the FETP to evaluate their relationship with renal function and histologic lesions in glomerular diseases. Thus, this study aims to assess the relationship between FETP and the clinicopathological findings and whether FETP predicts outcome in iMN.MethodThis study included patients with iMN that underwent kidney biopsies during the period from 2002 to 2020. We analyzed 24-h urinary protein excretion, FETP, and other clinicopathological findings at the kidney biopsy. The FETP was determined by the standard clearance technique based on 24-h urine collection: FETP = (urinary total protein / serum total protein) / (urinary creatinine / serum creatinine) × 100. A 30% decrease in eGFR or the occurrence of ESRD were the endpoints. The multivariate factors affecting the prognosis were analyzed with the Cox proportional-hazards model, and the cumulative risk of risk factors was analyzed by Kaplan‑Meier curve.ResultsA total of 153 subjects with MN were identified and were followed up for a median of 5.4 years. (age 64.9±13.6 [mean ± SD] years, male 73.2 %, hypertension 42.5 %, diabetes 10.5 %, nephrotic Syndrome 67.3 %, chronic kidney disease [CKD: eGFR<60ml/min/1.73m2] 51.9 %, eGFR 61.6±22.6 mL/min/1.73m2, urinary protein excretion [u-TP] 4.3±3.6 g/day, PCR 5.4±4.5 g/gCr, FETP 0.12±0.18 %, Selectivity Index [S.I] 0.23±0.39, fractional excretion of IgG [FEIgG] 0.065±0.170 %, glomerulosclerosis [GS] 13.9±13.7 %, interstitial fibrosis and tubular atrophy [IFTA] 12.2±10.0 %). FETP was more significantly associated with clinical parameters than PCR and FEIgG (Table.1). The high FETP group had a significantly worse renal prognosis during the follow-up periods than the low FETP group (Figure.1). Using Cox proportional hazards models, with FETP entered, and age, sex, eGFR,u-TP as covariates, FETP predict the primary endpoint with a hazards ratio of 0.343 (P<0.05).ConclusionThese results suggest that FETP would be superior to PCR, the standard measure of proteinuria, in predicting outcome in patients with iMN. FETP could indicate the increased glomerular protein permeability and decreased glomerular filtration function in iMN.MO267 Table 1:Transplant Kidney Biopsy Findings from Positive COVID-19 Cases

Therapeutic trials in adult FSGS: lessons learned and the road forward.

Focal segmental glomerulosclerosis (FSGS) is not a specific disease entity but a lesion that primarily targets the podocyte. In a broad sense, the causes of the lesion can be divided into those triggered by a presumed circulating permeability factor, those that occur secondary to a process that might originate outside the kidneys, those caused by a genetic mutation in a podocyte or glomerular basement membrane protein, and those that arise through an as yet unidentifiable process, seemingly unrelated to a circulating permeability factor. A careful attempt to correctly stratify patients with FSGS based on their clinical presentation and pathological findings on kidney biopsy is essential for sound treatment decisions in individual patients. However, it is also essential for the rational design of therapeutic trials in FSGS. Greater recognition of the pathophysiology underlying podocyte stress and damage in FSGS will increase the likelihood that the cause of an FSGS lesion is properly identified and enable stratification of patients in future interventional trials. Such efforts will facilitate the identification of effective therapeutic agents.

Central Leptin Receptor Blockade Restored Renal Glucose Handling in a Model of Estrogen Deficiency

Hormonal changes and estrogen deficiency accompanying menopause have several effects on women such as weight gain in the form of adipose tissue. An increase in adiposity associates with an increased production of leptin, an adipocyte derived hormone, that acts mainly in the central nervous system. Leptin regulates energy balance and plays a primary role in the regulation of glucose homeostasis. In this study, we aim to determine leptin's effect on renal glucose handling in an estrogen deficient adult female rat model (ovariectomized; OVX). We observed significant weight gain, increase in glomerular filtration rate (GFR, increase in creatinine clearance from 1.3 ml/min to 2.3 ml/min) and protein level of an early biomarker of kidney injury, the neutrophil gelatinase-associated lipocalin (NGAL, 90% increase in both glomeruli and tubules, measured by immunohistochemistry) in OVX rats when compared to their sham-operated controls. The mechanism proposed for the increase in GFR and renal damage is lipotoxicity due to intrarenal lipid accumulation associated with increased perirenal adipose tissue. Indeed, a significant accumulation of renal lipid, assessed using oil red O staining, was detected in the renal tubules of the OVX rats when compared with their controls. All these effects were partially reversed by treatment with a leptin receptor antagonist (LAN-6, 3µg/day), delivered for four weeks into the lateral ventricle by an osmotic pump. The sodium-glucose cotransporter-2 (SGLT2), the main mediator of renal glucose handling, is co-expressed in renal proximal tubules (PTs) with Na/H exchanger-3 (NHE3), a key player of salt regulation. Using immunohistochemistry, we measured significant reduction of renal SGLT2 protein expression level in OVX rats when compared with their controls. NHE3 protein expression level was also decreased in the same PTs. The decrease in SGLT2 and NHE3 protein expression measured in OVX rats and glycosuria detected in the urine of OVX rats was significantly reduced in rats treated with LAN-6. Serum glucose levels did not change significantly in the conditions under study. These findings suggest that in the absence of estrogen: 1. infiltration of renal lipid leads to renal injury, 2. kidney injury combines with an increase in GFR, 3. protein expression levels of SGLT2 and NHE3 are decreased and renal glycosuria develops. Finally, blocking activation of central leptin receptors in part restores kidney function. This may shed light in the understanding of leptin's regulation of glucose homeostasis and lead to potential benefits against kidney dysfunction caused by intrarenal lipid accumulation that develops during estrogen deficiency.

Quantification of Protein Reabsorption Energetics in the Kidney Proximal Tubule

Proteinuria can result from increased glomerular protein filtration, but insufficient or reduced protein reabsorption in the proximal tubule (PT) may also play a role. Protein is reabsorbed via receptor-mediated endocytosis, a process that is pH-dependent and requires energy in the form of ATP. Specifically, ATP is hydrolyzed by V-type ATPase, and the resulting energy is used to acidify the endocytic vesicle by pumping H+ ions into the endosome. This reduces the internal endosome pH and causes protein-receptor complex dissociation, which allows the protein to be further processed by the cell. Alterations in intracellular pH or ATP availability may impact the PT epithelial cells’ ability to form endosomes and reabsorb protein. To quantify the relationship between cellular pH, protein reabsorption, and ATP utilization, we developed a mathematical model that quantifies the energy demands of protein reabsorption in the kidney proximal tubule. The model assumes normal kidney function and focuses on albumin reabsorption for simplification. To determine the energy required to acidify a single endosome (ATP/endosome), we expanded upon a published mathematical model of general lysosome acidification that quantifies the H+ ions required to acidify a vesicle and the final pH reached, accounting for ClC-7 antiporters and proton leak, as a function of intracellular pH (Ishida et al. J Gen Phys 2013). The Nernst equation was used to determine the cumulative energy (ΔG) required to pump H+ ions into one endosome until a final stable internal pH is reached. To determine the rate of endosome formation required to reabsorb a normal filtered albumin load, we first calculated the albumin reabsorption rate per unit length of the proximal tubule as a function of albumin filtration rate, nephron number, and tubular dimensions. Next, using previous experimentally measured values for membrane internalization velocity and early endocytic radius, the rate of endosome formation required to reabsorb the filtered albumin load was calculated (total endosomes formed /sec). Total rate of ATP required to reabsorb a filtered load of albumin was then determined as the product of ATP/endosomes and total endosomes formed / second. At normal intracellular pH and normal filtered albumin load, it is estimated that 83,600 endosomes, with a radius of 0.34 µm, and 25,000 molecules of ATP are needed to reabsorb 740 µg of albumin per second for one proximal tubule. This is a very small fraction of total ATP utilized by the cell. To our knowledge, this model is the first to quantify the energetics of PT endosome formation and protein reabsorption. It provides a tool to quantitatively evaluate the impact of variations in factors such as intracellular pH, filtered protein load, and energy availability on the PT's ability to acidify endosomes and reabsorb protein. This may aid in future elucidation of pathological mechanisms of proteinuria.

Sheep urinary tract architecture is not affected by acute urethral obstruction

Urolithiasis affects the urinary tract of small ruminants, thereby requiring the animal to prematurely terminate breeding. Morphometric study of organs can be used as a diagnostic method. Thus, this study aimed to describe the macroscopic, histopathological, and histomorphometric changes in the urinary tract of sheep with urolithiasis. For this purpose, 14 healthy male Santa Inês sheep, approximately 90 days old, were studied and fed an experimental diet. After the development of urolithiasis, the animals were reorganized into two groups, D1 (without urolithiasis) and D2 (with urolithiasis) for comparative data analysis. Sheep were necropsied to evaluate the pathological changes, followed by macroscopic morphometric analysis, and the histopathological and histomorphometric characteristics were described. Urethral necrosis and a full urinary bladder were observed in all animals that developed the disease. The comparison between sheep with and without urolithiasis showed no significant difference (P &lt; 0.05) in the evaluated macroscopic morphometric variables, except for the right ureter width. Regarding the histopathological evaluation, multifocal areas of mild to moderate congestion within the glomerular tufts and protein in the tubular lumen of the kidneys were observed. In the liver, mild to moderate fatty degeneration was noted in the centrolobular regions, and an ulcerated focal area in the bladder mucosa was observed in only one animal. The present study demonstrated that the formulated diet was effective in inducing clinical disease. In acute obstructive urolithiasis in sheep tissue, lesions in the liver and urinary tract were observed, although there were no significant histomorphometric changes.

Thrombomodulin is upregulated in the kidneys of women with pre-eclampsia

The endothelial glycoprotein thrombomodulin regulates coagulation, vascular inflammation and apoptosis. In the kidney, thrombomodulin protects the glomerular filtration barrier by eliciting crosstalk between the glomerular endothelium and podocytes. Several glomerular pathologies are characterized by a loss of glomerular thrombomodulin. In women with pre-eclampsia, serum levels of soluble thrombomodulin are increased, possibly reflecting a loss from the glomerular endothelium. We set out to investigate whether thrombomodulin expression is decreased in the kidneys of women with pre-eclampsia and rats exposed to an angiogenesis inhibitor. Thrombomodulin expression was examined using immunohistochemistry and qPCR in renal autopsy tissues collected from 11 pre-eclamptic women, 22 pregnant controls and 11 hypertensive non-pregnant women. Further, kidneys from rats treated with increasing doses of sunitinib or sunitinib in combination with endothelin receptor antagonists were studied. Glomerular thrombomodulin protein levels were increased in the kidneys of women with pre-eclampsia. In parallel, in rats exposed to sunitinib, glomerular thrombomodulin was upregulated in a dose-dependent manner, and the upregulation of glomerular thrombomodulin preceded the onset of histopathological changes. Selective ETAR blockade, but not dual ETA/BR blockade, normalised the sunitinib-induced increase in thrombomodulin expression and albuminuria. We propose that glomerular thrombomodulin expression increases at an early stage of renal damage induced by antiangiogenic conditions. The upregulation of this nephroprotective protein in glomerular endothelial cells might serve as a mechanism to protect the glomerular filtration barrier in pre-eclampsia.

Effect of salt intake on residual renal function in rats receiving peritoneal dialysis

To assess the effect of salt intake on residual renal function in rats and explore the possible mechanism. SD rats were 5/6-nephrectomized to induce chronic renal failure followed by peritoneal dialysis for 4 weeks (n=18) or without dialysis treatment (control group; n=18). In both groups, the rats were divided into 3 subgroups and were given lowsalt diet (0.02% NaCl), normal salt diet (0.4% NaCl), and high-salt diet (4% NaCl). After 8 and 12weeks, blood pressure and creatinine and sodium levels in the blood, urine, and peritoneal dialysate of the rats were examined. Glomerular sclerosis, tubulointerstitial fibrosis, and protein expression levels of RAS components (ACE-1, AGT, and AT-1) in renal cortical tissue of the rats were evaluated. The residual renal function of the rats all decreased especially in rats with high salt intake for 8and 12 weeks. In peritoneal dialysis group, the rats with high-salt diet showed signficiantly increased renal interstitial fibrosis score (P=0.036), glomerular sclerosis index (P=0.045), systolic blood pressure (P=0.004), diastolic blood pressure (P=0.048), and renal expressions of AGT, ACE-1, and AT1 (P < 0.05) as compared with those with normal salt intake. In the rats fed the same high-salt diet, the renal interstitial fibrosis score, glomerular sclerosis index, diastolic blood pressure increase, and renal AGT and ACE-1 expression levels were significantly lower in the peritoneal dialysis group than in the control group (P < 0.05). A positive correlation was noted between the reduction of residual renal function and sodium intake in the rats. In rats with chronic renal failure, high salt intake promotes the activation of the renal RAS system, increases blood pressure, and agrevates renal fibrosis to accelerate the decline of residual renal function, and peritoneal dialysis partially reduces the damage of residual renal function induced by high-salt diets by removing excessive sodium.

Reduced Glomerular Endothelial Thrombomodulin Is Associated with Glomerular Macrophage Infiltration in Diabetic Nephropathy

The endothelial glycoprotein thrombomodulin regulates coagulation, inflammation, and apoptosis. In diabetic mice, reduced thrombomodulin function results in diabetic nephropathy (DN). Furthermore, thrombomodulin treatment reduces renal inflammation and fibrosis. Herein, thrombomodulin expression was examined in human kidney samples to investigate the possibility of targeting thrombomodulin in patients with DN. Glomerular thrombomodulin was analyzed together with the number of glomerular macrophages in 90 autopsied diabetic cases with DN, 55 autopsied diabetic cases without DN, and 37 autopsied cases without diabetes or kidney disease. Thrombomodulin mRNA was measured in glomeruli microdissected from renal biopsies from patients with DN and nondiabetic controls. Finally, glomerular thrombomodulin was measured in diabetic mice following treatment with the selective endothelin A receptor (ETAR) blocker, atrasentan. In diabetic patients, glomerular thrombomodulin expression was increased at the mRNA level, but decreased at the protein level, compared with nondiabetic controls. Reduced glomerular thrombomodulin was associated with an increased glomerular influx of macrophages. Blocking the ETAR with atrasentan restored glomerular thrombomodulin protein levels in diabetic mice to normal levels. The reduction in glomerular thrombomodulin in diabetes likely serves as an early proinflammatory step in the pathogenesis of DN. Thrombomodulin protein may be cleaved under diabetic conditions, leading to a compensatory increase in transcription. The nephroprotective effects of ETAR antagonists in diabetic patients may be attributed to the restoration of glomerular thrombomodulin.

Patients with Proliferative Lupus Nephritis Have Autoantibodies That React to Moesin and Demonstrate Increased Glomerular Moesin Expression.

Kidney involvement in systemic lupus erythematosus (SLE)—termed lupus nephritis (LN)—is a severe manifestation of SLE that can lead to end-stage kidney disease (ESKD). LN is characterized by immune complex deposition and inflammation in the glomerulus. We tested the hypothesis that autoantibodies targeting podocyte and glomerular cell proteins contribute to the development of immune complex formation in LN. We used Western blotting with SLE sera from patients with and without LN to identify target antigens in human glomerular and cultured human-derived podocyte membrane proteins. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified the proteins in the gel regions corresponding to reactive bands observed with sera from LN patients. We identified 102 proteins that were present in both the podocyte and glomerular samples. We identified 10 high-probability candidates, including moesin, using bioinformatic analysis. Confirmation of moesin as a target antigen was conducted using immunohistochemical analysis (IHC) of kidney biopsy tissue and enzyme-linked immunosorbent assay (ELISA) to detect circulating antibodies. By IHC, biopsies from patients with proliferative lupus nephritis (PLN, class III/IV) demonstrated significantly increased glomerular expression of moesin (p < 0.01). By ELISA, patients with proliferative LN demonstrated significantly increased antibodies against moesin (p < 0.01). This suggests that moesin is a target glomerular antigen in lupus nephritis.

Cathepsin L mediates glomerular endothelial cell injury by cleavaging complement C3 in trichloroethylene-sensitized mice

Objective: To observe the expressions of complement 3 (C3) and endothelial cell injury-associated proteins before and after cathepsin L (CTSL) blockade in renal injury of trichloroethylene (TCE) -sensitized mice. Methods: In June 2018, 41 SPF female BALB/c mice were divided respectively into blank control group (n=5) , vehicle control group (n=5) , TCE group (n=15) and TCE+CTSLi group (n=16) to establish trichloroethylene-sensitized mice model by pretreating the mice with intraperitoneal injection of CTSL inhibitor (CTSLi) and using TCE for the first and last challenge. According to the skin sensitization score, the mice were divided into positive group and negative group. 72 hours after the last challenge, the renal function indexes of the mice were detected, the pathological changes of mice kidneys were observed, and the glomerular C3 and endothelial cell damage-related proteins [vascular cell adhesion molecule 1 (VCAM-1) , tight junction protein 5 (Claudin-5) and Syndecan-1] expression levels were detected. Results: The sensitization rates of mice in TCE group and TCE+CTSLi group were 53.3% (8/15) and 50.0% (8/16) , respectively, and there was no significant difference between the two groups (P>0.05) . Compared with vehicle control group and the corresponding TCE negative group, the serum creatinine (CRE) and blood urea nitrogen (BUN) levels of mice in the TCE positive group was increased, while the TCE positive group were higher than the TCE+CTSLi positive group (P<0.05) . Pathological examination showed obvious vacuolar degeneration and cellular edema in the mice kidney of the TCE positive group. In the TCE+CTSLi positive group, the above pathological damage was significantly improved. Immunohistochemical results showed that the expression of glomerular C3 fragment and VCAM-1 in TCE positive group were significantly higher than that of the vehicle control and TCE negative group (P<0.05) , while TCE+CTSLi positive group was significantly lower than that of TCE positive group (P<0.05) . Western blot test results showed that the relative expression levels of Claudin-5 and Syndecan-1 protein in the mice glomeruli of TCE positive group were significantly lower than those in the vehicle control group and TCE negative group (P<0.05) . Compared with the TCE positive group, the Claudin-5 protein was increased in the kidney of the TCE+CTSLi positive group, but the difference was not statistically significant (P>0.05) , while the Syndecan-1 protein was significantly increased in the TCE+CTSLi positive group (P<0.05) . Conclusion: CTSL may mediate the glomerular structural damage by cutting complement C3, activating the complement system, damaging endothelial cell structural protein Syndecan-1 and overexpressing adhesion molecule VCAM-1 in TCE-sensitized mice. Inhibiting the expression of CTSL may be an effective way to protect the glomerular integrity of structure and function in pharmacology.

The ISN/RPS 2016 classification predicts renal prognosis in patients with first-onset class III/IV lupus nephritis

Lupus nephritis (LN) is a life-threatening complication of systemic lupus erythematosus. The 2003 pathological classification of LN was revised in 2016; it quantitatively evaluates the interstitium in addition to the glomeruli. We performed a retrospective multi-centre cohort study and investigated the utility of the 2016 classification—including the activity index (AI), chronicity index (CI), and each pathological component to predict complete remission or renal function decline, defined as 1.5-fold increase in serum creatinine levels—and compare with that of the 2003 classification. Ninety-one consecutive adult patients with first-onset class III/IV LN who were newly prescribed any immunosuppressants were enrolled and followed up for a median of 51 months from January 2004. Cox regression analysis demonstrated the subclasses based on the 2003 classification, which mainly evaluate glomerular lesions, were not associated with clinical outcomes. After adjustments for estimated glomerular filtration rate and urinary protein levels, higher CI and higher interstitial fibrosis and lower hyaline deposit scores were associated with renal functional decline. Similarly, higher CI and interstitial inflammation scores were associated with failure to achieve complete remission. Therefore, the 2016 classification can predict the clinical outcomes more precisely than the 2003 classification.

NPHS2 gene polymorphism aggravates renal damage caused by focal segmental glomerulosclerosis with COL4A3 mutation.

Focal segmental glomerulosclerosis (FSGS), a type of primary glomerular disease, is the leading cause of end-stage renal disease (ESRD). Several studies have revealed that certain single-gene mutations are involved in the pathogenesis of FSGS; however, the main cause of FSGS has not been fully elucidated. Homozygous mutations in the glomerular basement membrane gene can lead to early renal failure, while heterozygous carriers develop renal failure symptoms late. Here, molecular genetic analysis of clinical information collected from clinical reports and medical records was performed. Results revealed that nephrosis 2 (NPHS2) gene polymorphism aggravated renal damage in three FSGS families with heterozygous COL4A3 mutation, leading to early renal failure in index patients. Our findings suggest that COL4A3 and NPHS2 may have a synergistic effect on renal injury caused by FSGS. Further analysis of the glomerular filtration barrier could help assess the cause of kidney damage. Moreover, a detailed analysis of the glomerular basement membrane-related genes and podocyte structural proteins may help us better understand FSGS pathogenesis and provide insights into the prognosis and treatment of hereditary glomerulonephropathy.

Inhibition of neprilysin with sacubitril without RAS blockage aggravates renal disease in Dahl SS rats

Salt-sensitive (SS) hypertension is accompanied with severe cardiorenal complications. In this condition, elevated blood pressure (BP) resulting from salt retention is associated with counterintuitively lower levels of atrial natriuretic peptide (ANP). In plasma, ANP is degraded by the neprilysin; therefore, pharmacological inhibition of this metalloprotease (i.e., with sacubitril) can be employed to increase ANP level. We have shown earlier that sacubitril in combination with valsartan (75 μg/day each) had beneficial effects on renal function in Dahl SS rats. The goal of this study was to evaluate the effects of a higher dose of sacubitril on renal damage in this model. To induce hypertension, male Dahl SS rats were fed a 4% NaCl diet (HS) for 21 days, and were administered sacubitril (125 μg/day) or vehicle via s.c. osmotic pumps. At the end of the HS challenge, both groups exhibited similar outcomes for GFR, heart weight, plasma electrolytes, BUN, and creatinine. Sacubitril exacerbated kidney hypertrophy, but did not affect levels of renal fibrosis. We also observed aggravated glomerular lesions and increased formation of protein casts in the sacubitril-treated animals compared to controls. Thus, in Dahl SS rats, administration of sacubitril without renin-angiotensin-system blockage had adverse effects on renal disease progression, particularly in regards to glomerular damage and protein cast formation. We can speculate that while ANP levels are increased because of neprilysin inhibition, there are off-target effects of sacubitril, which are detrimental to renal function in the SS hypertensive state.

ВЛИЯНИЕ ЛОКАЛЬНОЙ АНТИГЕННОЙ СТИМУЛЯЦИИ МОЛОЧНОЙ ЖЕЛЕЗЫ НА ПОКАЗАТЕЛИ ИММУНОГЛОБУЛИНОВ В КРОВИ МЫШЕЙ

Immunoglobulins are large glomerular proteins in blood plasma and are an important factor in humoral immunity. The concentration of immunoglobulins in blood plasma directly characterizes the body's ability to protect itself from foreign agents. Immunoglobulins play an extremely important role during pregnancy and lactation. Immunoglobulins of classes A, M and G enter the body of newborns with colostrum, thus realizing the biological mechanism of protecting newborns from infectious environmental influences. The shortage of immunological components of colostrum by young animals is associated with the widespread prevalence of mastitis in livestock complexes, since any form of mastitis adversely affects the composition and sanitary and hygienic parameters of milk. This problem can be solved by local antigenic stimulation of the mammary gland with various thermostable antigens corresponding to the main causative agents of mastitis. For example, the treatment of the mammary glands with staphylococcal vaccine a few days before giving birth allows not only to protect the lactating herd from mastitis of staphylococcal etiology, but also makes it possible to transfer anti-staphylococcal immunity to young animals in the process of drinking colostrum. In our study, while continuing to study the physiological mechanisms of the implementation of the local immune response of the mammary gland, we wanted to record changes in the humoral factors of immunity upon local stimulation of the mammary gland with staphylococcal vaccine in an experimental model of lactating mice. The main purpose of this study was to com pare the concentration of immunoglobulins of classes A, M and G in blood plasma in lactating mice immunized with staphylococcal vaccine and individuals that were not immunized. According to the results of the experiment, data were obtained showing that the concentration of immunoglobulins of all studied classes in immunized individuals significantly exceeds the concentration of the corresponding immunoglobulins in mice of the control group.

A well-developed endolysosomal system reflects protein reabsorption in segment 1 and 2 of rat proximal tubules

Proteinuria is a well-established marker and predictor of kidney disease. The receptors megalin and cubilin reabsorb filtered proteins and thereby proteinuria is avoided. It is unknown if all segments of the proximal tubule are involved in clearing the filtrate or if there exists a reserve capacity in case of increased glomerular protein filtration. To determine this, we performed serial sectioning of rat kidney and used stereology to quantify the endolysosomal system of the three segments of cortical and juxtamedullary nephrons by electron microscopy. Immunohistochemistry was applied to analyze the adaptor protein Dab2, which assists in megalin mediated endocytosis, megalin, and endocytic uptake of two endogenous megalin ligands; retinol binding protein and β2-microglobulin at exact tubular positions. Proteinuric rats (puromycin-treated) and mice (podocin knock-out) were analyzed to clarify the response of the tubule to increased protein filtration. We found that the endolysosomal system was most prominent in segment 1 and 2, whereas segment 3 was less developed. The depth of ligand uptake varied among nephrons, but it descended into segment 2 although uptake was lower than in segment 1 and it was never observed in segment 3. This was supported by prominent expression of Dab2 in segment 1 and 2. When protein filtration increased, segment 3 was included in the reabsorption process in proteinuric animals. Thus, segment 1 and 2 are responsible for clearing the filtrate for protein during normal physiological conditions, but the tubule exhibits plasticity and is able to include segment 3 under proteinuric stress.

Nephroprotection by SGLT2i in CKD Patients: May It Be Modulated by Low-Protein Plant-Based Diets?

Sodium-glucose-transporter 2 inhibitors (SGLT2i) are a new class of anti-diabetic drugs that in large trials such as CREDENCE have shown also a reduction of glomerular hyperfiltration and albuminuria in type 2 diabetic patients. Hence, the interest toward SGLT2i is focused toward this potential nephroprotective effect, in order to reduce the progression to overt nephropathy, and it seems to be confirmed in the most recent DAPA-CKD trial. This is the reason why the indication for SGLT2i treatment has been extended to chronic kidney disease (CKD) patients with eGFR up to 30 ml/min, namely with CKD stage 1–3. In patients with CKD stage 3 to 5, the most recent KDIGO guidelines recommend low-protein diet and plant-based regimens to delay end-stage kidney disease (ESKD) and improve quality of life. Similarly to SGLT2i, low-protein diets exert renal-protective effects by reducing single nephron hyperfiltration and urinary protein excretion. Beyond the glomerular hemodynamic effects, both protein restriction and SGLT2i are able to restore autophagy and, through these mechanisms, they may exert protective effects on diabetic kidney disease. In this perspective, it is likely that diet may modulate the effect of SGLT2i in CKD patients. Unfortunately, no data are available on the outcomes of the association of SGLT2i and low-protein and/or vegan diets. It is therefore reasonable to investigate whether CKD patients receiving SGLT2i may have further advantages in terms of nephroprotection from the implementation of a low-protein and/or plant-based diet or whether this association does not result in an additive effect, especially in vascular nephropathies.