Tubulointerstitial Infiltration Research Articles

Cluster of differentiation-44 as a novel biomarker of lupus nephritis and its role in kidney inflammation and fibrosis.

CD44 is a transmembrane glycoprotein implicated in tissue inflammation and fibrosis. We investigated its role in kidney inflammation and fibrosis in a murine model of lupus nephritis (LN), and the clinico-pathological association of serum CD44 level in patients with biopsy-proven Class III/IV ± V LN. NZB/W F1 mice were treated with control IgG or anti-CD44 monoclonal antibody for 4 weeks and disease parameters assessed. Serum CD44 level in LN patients was determined by ELISA. Control groups included healthy subjects and patients with non-renal SLE or non-lupus renal disease. CD44 expression was absent in the normal kidney, but it was expressed in proximal and distal tubular epithelial cells and infiltrating cells in renal biopsies from patients with active proliferative LN. ScRNA-Seq datasets confirmed that CD44 was predominantly expressed in tubular cells and all immune cells identified in LN patients including tissue resident, inflammatory and phagocytic macrophages, Treg cells, effector and central memory CD4+ T cells, resident memory CD8+ T cells and naïve and activated B cells. Treatment of NZB/W F1 mice with anti-CD44 antibody preserved kidney histology and reduced proteinuria, tubulo-interstitial infiltration of CD3+, CD4+ and CD19+ immune cells, and mediators of kidney fibrosis compared to Control mice. Longitudinal studies showed that serum CD44 level increased prior to clinical renal flare by 4.5 months and the level decreased after treatment. ROC curve analysis showed that CD44 level distinguished patients with active LN from healthy subjects and patients with quiescent LN, active non-renal lupus, and non-lupus CKD (ROC AUC of 0.99, 0.96, 0.99 and 0.99 respectively). CD44 level correlated with leukocyte infiltration and interstitial inflammation scores in active LN kidney biopsies. Our findings suggest that CD44 plays a pathogenic role in renal parenchymal inflammation and fibrosis in active LN and monitoring CD44 may facilitate early diagnosis of flare.

Serum soluble interleukin-2 receptor alpha may predict tubulointerstitial inflammatory cell infiltration and short-term disease progression in immunoglobin A nephropathy.

This study aims to explore the relationship between serum soluble interleukin-2 receptoralpha (sIL-2Rα) levels and histologic features in immunoglobin A nephropathy (IgAN), and evaluate its predicting values on disease progression and remission status. IgAN patients were included retrospectively. Lee classification, Oxford classification and histological scoring were evaluated. Patients' estimated filtration rate (eGFR) and proteinuria remission status were collected during 6-month follow-up. Logistic regression was used to determine the risk factors and predicting value. Receiver operating characteristic (ROC) curve were used to determine the predicting value for outcome. One hundred seventy-two subjects were included in this study. Individuals in moderate-to-severe tubulointerstitial inflammatory cell infiltration group manifested with significantly elevated serum sIL-2Rα levels than those in non-to-mild group. Serum sIL-2Rα levels were positively correlated with infiltration scores. Serum sIL-2Rα was an independent risk factor for moderate-to-severe inflammatory cell infiltration [sIL-2Rα: OR 1.29 (1.015-1.640, p = 0.038)]. ROC curve analysis regarding predictive value for moderate-to-severe inflammatory cell infiltration of sIL-2Rα suggested area under curve was 0.859 (0.801-0.918, p = 0.000) when sIL-2Rα combined with eGFR < 60mL/(min·1.73 m2), 24-h proteinuria excretion > 1.0g, and hemoglobin. It showed good sensitivity (71.6%) and specificity (87.6%). Additionally, sIL-2Rα levels at kidney biopsy were strong predictive factor for kidney function loss 6months after kidney biopsy [OR 4.161 (1.013-17.088, p = 0.048)]. High serum sIL-2Rα was significantly associated with serious inflammatory cell infiltration in IgAN, and it showed strong predictive value for disease prognosis. Serum sIL-2Rα could be a useful noninvasive biomarker to evaluate the extent of histological injury and disease prognosis in IgAN.

Roles of M1 and M2 macrophage infiltration in post-renal transplant antibody-mediated rejection

BackgroundWe aimed to analyze the roles of M1 and M2 macrophage infiltration in post-renal transplant antibody-mediated rejection (AMR). MethodsA total of 102 recipients who underwent renal allotransplant from January 2020 to February 2023 were divided into an immune tolerance group (n = 56) and a rejection group (n = 46). The transplant renal biopsy specimens were harvested by ultrasound-guided puncture. The M1 and M2 macrophages in renal tissues were counted, and the M1/M2 ratio was calculated. The numbers of M1 and M2 macrophages and M1/M2 ratios in patients with different severities of interstitial fibrosis/tubular atrophy (IF/TA) and different degrees of tubulointerstitial inflammatory cell infiltration were compared. The predictive values of M1 and M2 macrophages and M1/M2 ratio for post-renal transplant AMR were clarified. ResultsThe rejection group had significantly more M1 and M2 macrophages and higher M1/M2 ratio than those of the immune tolerance group (P < 0.05). In the rejection group, infiltrating macrophages were mainly distributed in the glomerular and interstitial capillaries, with M1 macrophages being the predominant type. With increasing severity of IF/TA, the numbers of M1 and M2 macrophages and M1/M2 ratio rose in patients with post-renal transplant AMR (P < 0.05). The numbers and ratio had significant positive correlations with the levels of blood urea nitrogen and serum creatinine (P < 0.05). The areas under the curves (AUCs) of numbers and M1 and M2 macrophages and M1/M2 ratio for predicting post-renal transplant AMR were 0.856, 0.839 and 0.887, respectively. The combined detection had AUC of 0.911 (95% CI: 0.802–0.986), sensitivity of 90.43% and specificity of 83.42%. ConclusionsSignificant macrophage infiltration is present in the case of post-renal transplant AMR, and closely related to the severity of IF/TA and the degree of tubulointerstitial inflammatory cell infiltration.

#211 Low albumin levels are associated with intrarenal complement C4d deposits in critically ill patients with ANCA-associated renal vasculitides

Abstract Background and Aims Despite serum albumin levels being predictive for clinical outcome in ANCA-associated renal vasculitis, implications providing a direct link between low serum albumin levels and intrarenal lesions remain elusive. Therefore, we here aimed to systematically assess the clinical relevance of low albumin levels and scrutinize clinicopathological correlations to expand our current knowledge. Method We here retrospectively enrolled biopsy-proven cases of ANCA-associated renal vasculitis between 2015 till 2020 in a single-center observational study. Survival-curve analyses on long-term renal survival and short-term clinical recovery were performed. Correlative analyses between serum albumin levels, laboratory parameters, proteinuria levels, and histopathological lesions including tubulointerstitial immune cell infiltrates, lesions analogous to the Banff score and intrarenal complement deposition of C3c and C4d were performed. Results We here show that hypoalbuminemia below the median of 2.4 g/dL impairs long-term renal survival especially in MPO-positive ANCA-associated renal vasculitis (p = 0.006, HR: 5.0, 95% CI: 1.2-21.5), while short-term clinical recovery particularly in critically ill patients is negatively affected by low serum albumin levels (p = 0.0082, HR: 3.6, 95% CI: 1.1-11.7). Low albumin levels are associated with the urinary marker of tubular damage in the total cohort (β = −0.5, p = 0.01), PR3-ANCA (β = −0.5, p = 0.04) and MPO-ANCA (p = 0.002, β = −0.8), but not in critically ill patients (β = −0.5, p = 0.07) implying hypoalbuminemia to occur by extrarenal causation in the state of critical illness. We identified plasmacytic infiltrates to correlate with low albumin levels in the total cohort (β = −0.6, p = 0.004) and in the subgroup of critically ill patients (β = −0.7, p = 0.005). Banff-scored interstitial inflammation (i) was further observed to inversely correlate with albumin levels (β = −0.7, p = 0.02). Intrarenal C4d deposition showed a significant correlation with low serum albumin levels in the glomerular tuft (β = −0.4, p = 0.04) and in interstitial arteries (β = −0.7, p = 0.01). Conclusion In conclusion, long-term renal outcome is significantly affected by low serum albumin levels especially in the setting of MPO-ANCA seropositivity. By contrast, short-term recovery was predicted by low albumin levels in critically ill patients with ANCA-associated renal vasculitis. We here provide evidence that low levels of serum albumin might directly affect tubulointerstitial inflammation as reflected by plasmacytic immune-cell infiltration, and intrarenal C4d complement deposition in ANCA-associated renal vasculitis.

Prediction of treatment response in lupus nephritis using density of tubulointerstitial macrophage infiltration.

Lupus nephritis (LN) is a common disease with diverse clinical and pathological manifestations. A major challenge in the management of LN is the inability to predict its treatment response at an early stage. The objective of this study was to determine whether the density of tubulointerstitial macrophage infiltration can be used to predict treatment response in LN and whether its addition to clinicopathological data at the time of biopsy would improve risk prediction. In this retrospective cohort study, 430 patients with LN in our hospitalfrom January 2010 to December 2017 were included. We used immunohistochemistry to show macrophage and lymphocyte infiltration in their biopsy specimens, followed by quantification of the infiltration density. The outcome was the treatment response, defined as complete or partial remission at 12 months of immunosuppression. The infiltration of CD68+ macrophages in the interstitium increased in patients with LN. High levels of CD68+ macrophage infiltration in the interstitium were associated with a low probability of treatment response in the adjusted analysis, and verse vice. The density of CD68+ macrophage infiltration in the interstitium alone predicted the response to immunosuppression (area under the curve [AUC], 0.70; 95% CI, 0.63 to 0.76). The addition of CD68+cells/interstitial field to the pathological and clinical data at biopsy in the prediction model resulted in an increased AUC of 0.78 (95% CI, 0.73 to 0.84). The density of tubulointerstitial macrophage infiltration is an independent predictor for treatment response in LN. Adding tubulointerstitial macrophage infiltration density to clinicopathological data at the time of biopsy significantly improves risk prediction of treatment response in LN patients.

Sex differences in long-term kidney fibrosis following neonatal nephron loss during ongoing nephrogenesis

BackgroundClinical studies suggest that female sex plays a protective role in the development and progression of kidney disease. Recent experimental studies indicate that in male rats early nephron loss under ongoing nephrogenesis is accompanied by severe long-term sequelae. In humans, nephron formation occurs mainly in the third trimester, ceasing with 36 weeks of gestation. Due to perinatal complications, preterm infants delivered during this vulnerable period may undergo acute nephron loss. In rats nephrogenesis persists until postnatal day 10, reflecting the situation of human preterms with persisting nephrogenesis. In our animal model of neonatal uninephrectomy, female and male rats were uninephrectomized at day 1 of life. Hypothesizing sex-dependent differences, long-term renal outcome was assessed after 1 year.ResultsIn both sexes, neonatal uninephrectomy was not followed by arterial hypertension at 1 year of age. Compensatory weight gain and glomerular hypertrophy of the remaining kidney occurred in uninephrectomized female and male animals. Selected markers of interstitial inflammation and fibrosis were regulated sex-dependently. The expression of monocyte chemoattractant protein-1 was increased in females, while tubulointerstitial infiltration by M1 macrophages was significantly higher in males after neonatal uninephrectomy. Neonatally uninephrectomized male rats had more glomerulosclerosis and podocyte damage compared to females, which was assessed by a semiquantitative score and desmin staining. RT-PCR revealed that after neonatal uninephrectomy in the remaining contralateral kidney of female rats the expression of candidate genes of renal development and function, i.e., wt-1, nephrin, synaptopodin, gdnf, and itga8 was higher than in males.ConclusionsBased on these observations we conclude that female sex is protective in the long-term response of the kidney to acute nephron loss under active nephrogenesis.

Protease-Activated Receptor-2 and Phospholipid Metabolism Analysis in Hyperuricemia-Induced Renal Injury.

Interstitial inflammation is an important mechanism of pathological damage in renal injury caused by hyperuricemia. Protease-activated receptor-2 (PAR2) is a class of targets that act upstream of the PI3K/AKT/NF-κB pathway and is involved in various inflammatory diseases. We induced a hyperuricemia model in rats by adenine and ethambutol gavage in an in vivo experiment. We demonstrated that PAR2 and PI3K/AKT/NF-κB pathway expression were significantly upregulated in renal tissues, with massive inflammatory cell infiltration in the renal interstitium and renal tissue injury. Treating hyperuricemic rats with AZ3451, a selective metabotropic antagonist of PAR2, we demonstrated that PAR2 antagonism inhibited the PI3K/AKT/NF-κB pathway and attenuated tubular dilation and tubulointerstitial inflammatory cell infiltration. The phospholipid metabolism profiles provided a perfect separation between the normal and hyperuricemic rats. In addition, we also found that AZ3451 can affect phospholipid metabolism. Our work suggests that PAR2 may mediate hyperuricemia-mediated renal injury by activating the PI3K/AKT/NF-κB pathway. The PAR2 antagonist AZ3451 may be a promising therapeutic strategy for hyperuricemia-induced inflammatory responses.

#6227 SPONTANEOUS REMISSION OF MEMBRANOUS VARIANT OF PGNMID WITH MONOCLONAL IGG2κ

Abstract Background and Aims The clinical significance of the morphological patterns of glomerular injury and of each IgG subclass in proliferative glomerulonephritis (GN) with monoclonal immunoglobulin deposits (PGNMID) is not well understood. Literature suggests that PGNMID with certain histological features such as membranous or mesangio-proliferative features or non-IgG3 subclass staining may be associated with a more favourable renal prognosis. We present a patient with membranous variant of PGNMID with monoclonal IgG2κ who had spontaneous remission without recurrence over a 2-year follow-up. Method Not applicable Results A 62-year-old Malay gentleman presented with new onset nephrotic range proteinuria, on a background of type 2 diabetes mellitus with diabetic duration of 5 years, hypertension and hyperlipidemia. He was mildly hypertensive (blood pressure 148/87) and non-edematous at presentation. Investigations revealed preserved kidney function [serum creatinine (sCr) 66µmol/L], bland urinalysis, and 24-hour urinary total protein (UTP) of 3.74g/day. Serum albumin (sAlb) was 35g/L. Serum complements were not low. Autoimmune markers inclusive of anti-double stranded DNA antibodies, anti-neutrophil cytoplasmic antibodies, and anti-phospholipase A2 receptor (PLA2R) antibodies were negative. Viral serologies were also unremarkable. No monoclonal band was detected on serum electrophoresis, while serum immunofixation was not performed. Kidneys were normal sized. Renal biopsy performed demonstrated 28 glomeruli, of which only 1 was globally sclerosed. Glomeruli capillary walls were diffusely thickened by vacuolations. Capillary loops were mostly single contoured although occasional focal double contouring was seen. Masson trichome stain revealed fuchsinophilic subepithelial immune deposits while periodic acid methenamine silver stain showed argyrophilic basement membrane spikes. Variable mesangial expansion and hypercellularity was observed, as well as mesangiolysis. Focal leukocyte margination was present, but significant endocapillary proliferation was not seen. There were no Kimmelstiel-Wilson lesions. Tubular atrophy with interstitial fibrosis was overall mild (10% of parenchymal area) and there were no tubulointerstitial infiltrates. Immunofluoresence showed granular staining of IgG (1-2+), C3 (2-3+) along glomerular capillary walls, as well as presence of kappa light chain restriction (κ 1-2+; λ negative in glomeruli). Immunostaining for PLA2R was negative. IgG subclass analysis performed in view of kappa restriction revealed isolated deposition of IgG2. Electron microscopy was not available. Overall findings were most consistent with membranous variant of PGNMID with monoclonal IgG2κ. Age-appropriate and symptom-directed malignancy screening did not reveal significant abnormalities. Given that the patient was minimally symptomatic, a trial of conservative therapy was initiated with optimized non-immunosuppressive anti-proteinuric therapy using renin-angiotensin-aldosterone and sodium-glucose co-transporter-2 inhibitors. Gradual spontaneous remission was observed without recurrence over 2 years of follow-up (sCr 77µmol/L; sAlb 40g/L; 24-hour UTP 0.22g/day). Conclusion Our case adds to the literature that a membranous variant of PGNMID with monoclonal IgG2κ may be associated with better renal outcomes. Despite the lack of proliferative changes, it is possible that membranous variants may share similar underlying disease mechanisms as the other proliferative variants. Large international registries to allow the correlation of morphological features and IgG subclasses with clinical outcomes are required to confirm our observation.

#4015 RECOMBINANT HIGH-DENSITY LIPOPROTEIN MODULATES INFLAMMATORY RESPONSE AND RENAL DYSFUNCTION IN A SWINE MODEL OF SEPSIS-INDUCED ACUTE KIDNEY INJURY

Abstract Background and Aims Sepsis is a severe and dysregulated inflammatory disease that often precedes the development of acute kidney injury (AKI) with consequent worsening outcome. Although clinical data demonstrate that high-density lipoprotein (HDL) levels drop in septic patients with a poor prognosis, little is known about the molecular basis of HDL's role in systemic inflammation and renal function. Here we investigate the possible effects of a novel engineered HDL-mimetic (CER-001) in a swine model of lipopolysaccharide (LPS)-induced AKI. Method Sepsis was induced by intravenous infusion of a saline solution containing 300 μg/kg of LPS in a porcine model. The animals were randomized into three groups: LPS (endotoxemic pigs, n = 6), CER20 (endotoxemic pigs treated with a single dose of CER-001 20mg/kg; n = 6), and CER20 × 2 (endotoxemic pigs treated with two doses of CER001 20mg/kg; n = 6). Animals were sacrificed after 24h from the start of experimental procedure. Renal histologic and biochemical changes were analyzed. Endothelial dysfunction biomarkers, circulating pro-inflammatory mediators, LPS and Apolipoprotein A-I (Apo A-I) levels were quantified with ELISA assay. Systemic complement activation was evaluated by Wieslab kit. Results Untreated animals were highly susceptible to LPS challenge and usually succumbed before completing the study protocol (LPS group 16.7%). CER-001 treatment increased the survival rate of endotoxemic pigs, compared to the LPS group (CER20, 50%; CER20 × 2, 66.7%). Furthermore, as shown in Figure 1A, LPS injection led to a time-dependent increase of IL-6 in endotoxemic animals respect to basal condition (T0). CER-001 treatment was able to reverse LPS effects. In particular, the second infusion of CER-001 three hours after the first dose (T3) strongly reduced IL-6 serum levels back to basal level (LPS p &amp;lt;0.05). Similarly, we found high levels of TNF-α and MCP-1 in endotoxemic pigs that were significantly decreased in both CER-001 treatment arms (T24, CER20 × 2 vs LPS group, IL-6, p = 0.0086; TNF-α, p&amp;lt;0.0001; MCP-1, p = 0.0009). In addition, CER-001 treatment ameliorated systemic endothelial dysfunction by reducing VCAM and ICAM serum levels. A significant activation of classical and alternative complement pathway (vs T0 p &amp;lt;0.05) was observed at 1h, 3h and 24h after LPS infusion. CER001 treatment significantly prevented systemic complement activation in both treated groups (vs LPS p &amp;lt;0.05). We also investigated whether CER001 infusions significantly prevented renal tissue damage. Endotoxemic pigs presented oliguric AKI with increased tubulo-interstitial infiltrate, extensive collagen deposition, and glomerular thrombi; CER-001 treatment preserved renal parenchyma, recovered urine output, decreased creatine levels, and reduced the biomarkers of tubular damage, Cystatin C and KIM-1, both in serum and urine samples. Considering that HDL has a very high affinity for LPS, we evaluated the circulating LPS concentration in treated animals. We observed that LPS levels were greatly reduced in treated animals and the effects are more evident after the second infusion of CER-001(Figure 1B). Therefore, we also examined LPS levels in bile samples and we observed a dose-dependent increasing amount of endotoxin in the CER001 treated septic pigs. Conclusion This preclinical data indicates that CER001 treatment prevents systemic inflammation thereby limiting renal damage. The mechanism of action is two-fold consisting of both the scavenging of endotoxin and a direct anti-inflammatory effect of CER-001.

#4544 THE ASSOCIATION OF TAMOXIFEN TO THE CONSERVATIVE CKD TREATMENT PROMOTED ADDITIONAL ANTIFIBROTIC EFFECTS IN A MODEL OF HYPERTENSIVE NEPHROSCLEROSIS

Abstract Background and Aims Chronic kidney disease (CKD) is an insidious, progressive and highly debilitating condition, which leads to the loss of renal function and to the need of life-sustaining renal replacement therapy. The conservative treatment of CKD is mainly based in the blockade of the renin-angiotensin-aldosterone system (RAAS), which can be associated to immunosuppressant drugs, according to the etiology of the renal injury. However, this pharmacological approach is not able to prevent CKD progression completely. Until the present moment, medical community still lack of a specific drug to effectively stop the progression of renal fibrosis associated to CKD, which once triggered became irreversible. Tamoxifen (TAM) is an estrogen receptor antagonist, widely employed for the clinical treatment of breast cancer, and responsible for saving a number of lives worldwide, in the past decades. This well-tolerated and cost-effective drug have been reported to exert antifibrotic effects in both experimental and human peritoneal fibrosis. Moreover, our research group already demonstrated that TAM efficiently prevented albuminuria, glomerulosclerosis, and interstitial fibrosis in a model of CKD. In the present study, we investigated if the association of TAM to the classic conservative treatment of CKD, here obtained by the association of Losartan (LOS) and Micofenolate Mofetil (MMF), could promote further renoprotection in an experimental model of hypertensive nephrosclerosis, induced by the chronic nitric oxide synthase blockade, obtained by the oral administration of L-NAME (NAME), associated to a high sodium (HS) diet, in rats. Method The experimental protocol was approved by the local Research Ethics Committee (CAPPesq) and was developed in strict conformity with the international standards for care and manipulation of laboratory animals. Thirty male Wistar rats were kept with a 3.2% HS diet for 15 days of adaptation, before the protocol start. After this period, the animals continued to receive the HS diet and were divided among 5 groups. CONT: receiving no further drugs or treatments, NAME: treated with 70 mg/kg/d of L-NAME, diluted in drinking water, LOS: NAME rats treated with 50mg/Kg/d of LOS, also diluted in drinking water, MMF: NAME rats treated with 10 mg/Kg/d of MMF given by gavage, TAM: NAME rats treated with 10 mg/Kg/d also by gavage, and LOS+MMF+TAM: NAME rats treated with all the drugs simultaneously. Systolic blood pressure (SBP), urinary albumin excretion (24 h uAE), glomerulosclerosis (GS), glomerular ischemia (GI), interstitial fibrosis (INT), tubulointerstitial infiltration of macrophages (CD68) and T-cells (CD3), as well as renal cortical interstitial collagen I (COLL1) and fibronectin (FIBRO) accumulation were evaluated after 30 days of treatment. Results The association of TAM to the classic treatment of CKD improved the renoprotection obtained by LOS+MMF. The triple combined treatment significantly reduced hypertension, albuminuria, glomerular structural damage, renal macrophage and T-cell infiltration in NAME rats, compared to the animals treated with the respective monotherapies. Moreover, both TAM alone or the association, were equally effective in reducing interstitial collagen and fibronectin accumulation, in NAME rats. Data are presented as Mean ± SE. For One-way ANOVA statistical analysis, we considered: p&amp;lt;0.05 vs.*CONT, #NAME, †LOS, §MMF, &amp;TAM. Conclusion Our preliminary results shown TAM to be effective in reducing renal inflammation and fibrosis in the chronic nitric oxide synthase blockade model and to exert additional renoprotective effects when associated to LOS and MMF in all the analyzed parameters. Although further studies with different nephropathy models are still required in order to confirm our findings, here we suggest TAM to be a potential adjuvant in the conservative management of CKD.

Pembrolizumab induced-C3 glomerulonephritis and RBC cast nephropathy: a case report

BackgroundImmune checkpoint inhibitors (ICIs) are increasingly being used in the treatment of several cancers. Pembrolizumab is an anti-programmed cell death-1 (anti-PD-1) monoclonal antibody that is approved for the treatment of metastatic non-small cell lung cancer (NSCLC). Pembrolizumab-associated renal toxicity is relatively rare, even in pembrolizumab-associated glomerulonephritis. In this study, we report a rare case of pembrolizumab-induced C3 glomerulonephritis (C3GN) and RBC cast nephropathy.Case presentationA 68-year-old man with NSCLC was receiving treatment with pembrolizumab. After 19 cycles of pembrolizumab therapy, he presented with gross hematuria, severe lower-limb edema and oliguria. Laboratory tests revealed hypoalbuminemia, increased serum creatinine and low serum C3 level. Renal biopsy revealed a typical membranoproliferative glomerulonephritis accompanied by remarkable RBC casts in tubular cavities and tubulointerstitial infiltration of CD8-positive lymphocytes. Based on C3-only immunofluorescence deposit on glomeruli, a diagnosis of C3GN was made. Pembrolizumab was considered the cause of C3GN. Pembrolizumab was discontinued immediately, and 60 mg/day of prednisone was initiated. One dose of cyclophosphamide (400 mg, IV) was also administered. Upon treatment, his symptoms improved rapidly and serum creatinine decreased a lot. However, the patient became dialysis dependent eventually.ConclusionThis is the first case of C3GN with RBC cast nephropathy caused by ICIs. This rare case caused by the prolonged use of pembrolizumab further strengthens the relationship between ICIs and C3GN. Thus, periodic evaluation of urine and renal function is recommended in patients receiving pembrolizumab and other ICIs.

Kidney involvement of chronic active Epstein-Barr virus infection.

A 54-year-old Japanese woman presented with a fever of unknown origin and renal dysfunction. She had experienced intermittent fever for 10 years, but the frequency increased to a constant fever. Her renal function had gradually deteriorated over the past year, and serum creatinine and urinary α1-microglobulin levels were elevated to 2.23 mg/dL and 113 mg/L, respectively. Physical and laboratory examinations were unremarkable, except for mild anemia and elevated serum creatinine levels. Analysis of peripheral blood lymphocyte subsets showed 59% CD3, 35% CD4 and 31% CD8. Epstein-Barr virus (EBV) serology showed elevated levels of EBV capsid antigen IgG and EBV nuclear antigen (1:1280 and 1:40, respectively). Plasma EBV-DNA level was 5.63 log IU/mL, and an oligoclonal band was detected by Southern blot hybridization of EBV terminal repeat analysis. Quantification of EBV-DNA in peripheral lymphocyte subset using magnetic beads showed that EBV predominantly resided in CD4+ T-cells. (CD4, 258,768; CD8, 27,738; and CD56, 23,369; IU/µgDNA, respectively). T-cell receptor α gene rearrangement was undetected. Based on the above, she was diagnosed with chronic active EBV infection (CAEBV). 18F-fluorodeoxyglucose positron emission tomography/computerized tomography showed bilateral kidney enlargement and mildly enhanced uptake (maximum standardized uptake value 4.08 g/mL) (Figure 1 top left). Kidney biopsy specimen stained with hematoxylin and eosin showed diffuse tubulointerstitial infiltration of small to medium-sized lymphocytes in the renal parenchyma (Figure 1 top right). Massive infiltration of CD4+ T-cells was documented by immunohistochemical staining (Figure 1 bottom left). Furthermore, EBV-infected cells were detected by in situ hybridization with EBV-encoded RNA (EBER) (Figure 1 bottom right). Immunostaining of EBV nuclear antigen-2 and latent membrane protein 1 were negative. EBER-positive cells were distributed in the same region as CD4+ T-cells, consistent with the predominant presence of EBV-DNA in CD4+ T-cells. Her renal function had improved after initiating chemotherapy, and she is scheduled for hematopoietic stem cell transplantation. The clinical features of EBV infection vary according to the type of EBV-infected cells. The predominant EBV-infected cells in EBV-induced hemophagocytic lymphohistiocytosis are CD8+ T cells, whereas in CAEBV, CD4+ T cells are predominantly infected. Although rare, renal invasion of EBV should be considered in cases with renal impairment, and its clinical significance needs to be investigated. AF and SS wrote and edited the manuscript, provided patient care and reviewed the literature. KS and MT provided patient care. TS and KM wrote and edited the manuscript. All authors reviewed and approved the final version of the manuscript We thank Dr. Yuka Torii (Department of Pediatrics, Nagoya University, Japan) for quantification of the EBV viral load of lymphocyte subsets using magnetic beads. We also thank Dr. Hiroshi Kimura (Department of Virology, Nagoya University, Japan) for his helpful advice on patient care. We appreciate the staff of the Department of Hematology, Kameda Medical Center for providing patient care. We thank Editage (www.editage.jp) for English language editing. The authors declare they have no conflicts of interest. The authors received no specific funding for this work. This study was conducted according to the Declaration of Helsinki, and written consent was obtained from the patient for this report. The patient provided written informed consent for the publication of this case, with the removal of all identifying information to ensure anonymity and maintain privacy. The data that support the findings of this study are available from the corresponding author upon reasonable request. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Leukocyturia and hematuria enable non-invasive differentiation of Bowman’s capsule rupture severity in PR3-ANCA glomerulonephritis

BackgroundRenal involvement is a common and severe complication of anti-neutrophil cytoplasmic antibody-(ANCA)-associated vasculitis potentially resulting in pauci-immune necrotizing and crescentic ANCA glomerulonephritis (GN) with rapid deterioration of kidney function, progression to end stage kidney disease or, if left untreated, lethal exitus. Analysis of the urinary sediment routinely supports clinical management of ANCA GN, but histopathological implications of aberrancies in the urinary sediment mostly remain elusive. Therefore, we aimed to systematically assess the correlation of aberrancies in the urinary sediment and clinico-pathologic findings.MethodsA total of 42 kidney biopsies with ANCA GN were retrospectively analyzed in a single-center observational study. Laboratory and histopathological parameters were systematically analyzed and correlated with findings of the urinary sediment.ResultsIn the overall ANCA GN cohort, leukocyturia and hematuria were associated among each other, and with markers for non-selective glomerular damage, respectively. Non-invasive measurement of leukocyturia indicated focal (but not extensive) Bowman’s capsule rupture (BCR) specifically in proteinase-3 (PR3)-ANCA GN, whereas hematuria correlated with extensive (but not focal) BCR. Concerning intrarenal immune cell infiltration, leukocyturia was associated with tubulointerstitial plasma cell infiltration in PR3-ANCA GN. Finally, none of these associations were detectable in myeloperoxidase-ANCA GN, implying different modes of kidney damage.ConclusionWe herein expand our current knowledge by providing evidence that leukocyturia and hematuria enable non-invasive differentiation of BCR severity specifically in PR3-ANCA GN.Graphical abstract

Tubulointerstitial B-cell infiltration and tertiary lymphoid tissue in adult-onset immunoglobulin A vasculitis with nephritis.

This study aimed to examine tubulointerstitial B-cell infiltration in patients with adult-onset immunoglobulin A vasculitis (IgAV) and nephritis (IgAV-N), and to evaluate whether B-cell infiltration correlated with clinicopathological variables at kidney biopsy and with short-term renal outcomes. Twenty patients with adult-onset IgAV-N and 10 control patients with thin basement membrane nephropathy (TBMN) were retrospectively examined. The lymphatic organization was graded based on B-cell infiltration and was classified into 4 groups: 0-T cells without B cells, 1-scattered B and T cells, 2-clustered B and T cells, and 3-nodular compartmentally arranged B- and T-cell aggregates, equivalent to tertiary lymphoid tissue (TLT). The B-cell infiltration grade was significantly higher in patients with IgAV-N than in patients with TBMN, and no age differences were observed. The B-cell infiltration grade in patients with IgAV-N was significantly correlated with age, serum IgA level, renal dysfunction, and tubulointerstitial injury parameters, but was not correlated with duration after purpura or glomerular injury parameters. Most patients with IgAV-N were treated with corticosteroids. The proteinuria level was significantly decreased, but renal function was not improved in 12 patients after the 24-month follow-up compared with the values at baseline. The B-cell infiltration grade was significantly correlated with renal dysfunction after 24months of follow-up. The B-cell infiltration grade in patients with IgAV-N was associated with renal dysfunction and tubulointerstitial injuries but not with glomerular injury parameters. B-cell infiltration and TLT might have a pathologically significant role in irreversible renal dysfunction in patients with early phase adult-onset IgAV-N.

Levels of donor-derived cell-free DNA and chemokines in BK polyomavirus-associated nephropathy.

BK polyomavirus-associated nephropathy (BKPyVAN) carries a risk of irreversible allograft injury. While detection of BK viremia and biopsy assessment are the current diagnostic gold standard, the diagnostic value of biomarkers reflecting tissue injury (donor-derived cell-free DNA [dd-cfDNA]) or immune activation (C-X-C motif chemokine ligand [CXCL]9 and CXCL10) remains poorly defined. For this retrospective study, 19 cases ofBKPyVANwere selected from the Vienna transplant cohort (biopsies performed between 2012 and 2019). Eight patients with T cell-mediated rejection (TCMR), 17 with antibody-mediated rejection (ABMR) and 10 patients without polyomavirus nephropathy or rejection served as controls. Fractions of dd-cfDNAwere quantified using next-generation sequencing and CXCL9 and CXCL10 were detected using multiplex immunoassays. BKPyVANwas associated with a slight increase in dd-cfDNA(median; interquartile range: .38% [.27%-1.2%] vs. .21% [.12%-.34%] in non-rejecting control patients; p=.005). Levels were far lower than in ABMR (1.2% [.82%-2.5%]; p=.004]), but not different from TCMR (.54% [.26%-3.56%]; p=.52). Within theBKPyVANcohort, we found no relationship between dd-cfDNAlevels and the extent of tubulo-interstitial infiltrates,BKPyVANclass and BK viremia/viruria, respectively. In some contrast to dd-cfDNA, concentrations of urinary CXCL9 and CXCL10 exceeded those detected in ABMR, but similar increases were also found in TCMR. BKPyVANcan induce moderate increases in dd-cfDNAand concomitant high urinary excretion of chemokines, but this pattern may be indistinguishable from that of TCMR. Our results argue against a significant value of these biomarkers to reliably distinguishBKPyVANfrom rejection.

Clinicopathological significance of tubulointerstitial CD68 macrophages in proliferative lupus nephritis.

Tubulointerstitial macrophage plays a pathogenic role in renal damage of lupus nephritis (LN). However, the clinical and pathological role of these CD68 macrophages has not been fully described. The aim of the present study is to decipher the correlation between clinicopathological features and tubulointerstitial CD68 macrophages in 76 proliferative LN patients and further evaluate the prognostic significance of tubulointerstitial CD68 macrophages. Tubulointerstitial CD68 macrophages were quantitated by standard histochemical staining. The correlation between the number of tubulointerstitial CD68 macrophages and clinicopathological features was analyzed by Spearman's correlation analysis. Factors potentially affecting renal prognosis were further evaluated by Cox regression analysis. Among the 76 proliferative LN cases, the number of CD68 macrophage infiltrates was positively correlated with serum creatinine (SCr) level, the proportion of glomeruli sclerosis and focal segmental sclerosis, tubulointerstitial inflammation, and chronicity indices, while negatively correlated with the glomerular filtration rate. During a mean follow-up period of 45months, 5 patients (6.6%) progressed to dialysis, and 3 patents (3.9%) had a twofold increase in SCr. Multivariate Cox regression analysis showed that the number of tubulointerstitial CD68 macrophages was an independent variable associated with poor renal outcomes (HR = 1.002, P = 0.012). The optimal cutoff value of tubulointerstitial CD68 macrophages was 340/mm2 in our study with 87.5% sensitivity and 61.8% specificity to predict end-stage renal disease within 4years. The number of tubulointerstitial CD68 macrophages was positively linked to poor prognosis of LN. Urgent immunosuppression should be considered in LN patients with abundant tubulointerstitial CD68 macrophages. Key Points • Tubulointerstitial CD68 macrophage infiltrates are positively correlated with clinicohistologic impairment in proliferative lupus nephritis. • The positive association between the number of tubulointerstitial CD68 macrophages and poor renal outcome of lupus nephritis patients were observed. • Urgent immunosuppression and monitor are required when abundant tubulointerstitial CD68 macrophage infiltrates are detected.

MO054: Histological Findings in Drug-Induced Acute Interstitial Nephritis

Abstract BACKGROUND AND AIMS Acute interstitial nephritis (AIN) represents an important cause of acute kidney injury. The most common etiology of AIN is drug-induced disease. Definitive diagnosis of AIN requires renal biopsy. Despite this, there is a lack of consensus regarding precise histological diagnostic criteria, and the clinical significance of common histological findings is uncertain. The aim of this study was to explore the histological characteristics of drug-induced AIN and to evaluate their impact on the renal outcome. METHOD We conducted a retrospective study that included patients with biopsy proven drug-induced AIN, followed in the department of nephrology of Charles Nicolle hospital in Tunis during the period between 1990 and 2019. Histological diagnosis of AIN was based on the presence of an interstitial inflammatory cell infiltrate with or without fibrosis. RESULTS During the period of the study, we collected 69 cases of biopsy proven AIN. The diagnosis of drug-induced AIN was performed in 23 patients (33%), with a medium age of 35 years. In eight patients (35%), nonsteroidal anti-inflammatory drugs were the culprit, followed by antibiotics in six patients (26%) and other drugs in three cases (13%). In six patients (26%), no single drug could be identified as the culprit, and these were classified as multidrug causes. The presence of acute kidney disease was the main indication for the kidney biopsy. All cases were characterized by a prominent tubulointerstitial inflammatory cell infiltrate. Interstitial infiltration was mild in three cases (13%), moderate in five cases (17%) and severe in 15 cases (65%). The interstitial infiltrate consisted predominantly of lymphocytes. The presence of eosinophils has been noted in 10 cases (43%). Interstitial granulomas were noted in two patients (8%). Interstitial edema and tubulitis were observed in 13 (56%) and 6 cases (26%), respectively. In seven patients (30%) interstitial fibrosis was present, and four patients (17%) had mild-to-moderate tubular atrophy, whereas tubular necrosis was present in seven cases (30%). After a mean follow up of 24 months, eight patients (35%) progressed to chronic kidney disease. Histological factors associated with poor renal outcome, were interstitial fibrosis (P ˂ 0.001) and tubular atrophy (P = 0.01). Patients with interstitial edema (P ˂ 0.001) or tubulitis (P = 0.005) had a better renal prognosis. CONCLUSION Renal biopsy is required to establish a definitive diagnosis of drug-induced AIN. Nevertheless, it is not carried out in a systematic way. The composition of cells in the interstitial infiltrate may at times be helpful in determining the etiology of AIN. In particular, the presence of a considerable proportion of eosinophils favors a diagnosis of drug-induced AIN. Moreover, during drug-induced AIN, histological data have a great prognostic value. In our study, the main histological prognostic factors were interstitial fibrosis, tubular atrophy, interstitial edema and tubulits. Further studies are needed to validate these results and establish a score based on histological data in order to predict renal outcome during drug-induced AIN.

Iguratimod alleviates tubulo-interstitial injury in mice with lupus

Introduction Tubulo-interstitial injury is a poor prognostic factor for lupus nephritis (LN). Here, we tested whether iguratimod could inhibit tubulo-interstitial injury in LN. Methods MRL/lpr mice, an animal model of lupus, were treated with iguratimod or vehicle solution. Pathological changes of kidney were evaluated blindly by the same pathologist. Renal type I collagen (COL-I), IgG, E-cadherin, fibroblast-specific protein 1 (FSP-1) were detected by immunofluorescence, immunohistochemical staining or quantitative real-time PCR. After treated with transforming growth factor β1 (TGF-β1) and iguratimod, E-cadherin, fibronectin, Smad2/3, p38 MAPK, p-Smad2/3, and p-p38 MAPK, β-catenin and TGF-β type II receptor (TGFβRII) in HK2 cells were measured by western blotting, quantitative real-time PCR or immunofluorescence. Results Iguratimod reduced immune deposition along the tubular basement membrane, inhibited the tubulo-interstitial infiltration of inflammatory cells, and alleviated tubular injury in MRL/lpr mice. Moreover, Iguratimod eased the tubulo-interstitial deposition of collagen fibers, which was confirmed by decreased expression of COL-I. Furthermore, iguratimod suppressed the expression of FSP-1 and increased that of E-cadherin in renal tubular epithelial cells. In HK2 cells cultured with TGF-β1, iguratimod treatment not only reversed cellular morphological changes, but also prevented E-cadherin downregulation and fibronectin upregulation. In addition, iguratimod inhibited phosphorylation of TGFβRII, Smad2/3 and p38 MAPK in HK2 cells treated with TGF-β1, and also blocked nuclear translocation of β-catenin. Conclusion Iguratimod eased tubulo-interstitial lesions in LN, especially tubulo-interstitial fibrosis, and might have potential as a drug for inhibiting the progression of tubulo-interstitial fibrosis in LN.

IDF21-0279 Association between Renal Tubulointerstitial Inflammatory Cells Infiltration and Renal Outcome in Diabetic Nephropathy

Background: Diabetic nephropathy (DN) continues to be the leading cause of end-stage renal disease (ESRD) worldwide, and increased amount of evidence supports that inflammation plays an important role in DN progression.

Establishment of an Animal Model of Vesicoureteral Reflux Renal Injury through Partial Bladder Outlet Obstruction

To establish an animal model of reflux renal damage through bladder outlet obstruction. Sixty male C57BL/6 mice aged 6-8 weeks were randomly assigned to a control group, a sham operation group, and a partial bladder outlet obstruction (PBOO) group, with 20 mice in each group. Laparotomy were performed on the PBOO mice under anesthesia in order to separate the bladder necks and to perform guided partial ligation of the bladder neck with a metal rod of 0.3 mm diameter. Mice in the sham operation group had laparotomy and had their bladder necks separated without ligation. The control group did not receive any treatment. 7 days after the surgery, 12 surviving mice were randomly selected from each group to observe the general changes of the bladder, ureter, renal pelvis and kidney. Retrograde urography was performed through the bladder. Kidney tissues were extracted for histopathological analysis. The expression levels of Vimentin, proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (α-SMA) were examined with Western blot, immunohistochemistry and immunofluorescence staining tests, respectively. Compared with the control and sham operation group, the bladder, ureter, and renal pelvis of the mice in the PBOO group were significantly enlarged, vesicoureteral reflux was more obvious, the kidney volume and mass increased ( P<0.001), and renal parenchyma became thinner ( P<0.000 1). Histopathological staining showed glomerular atrophy, renal tubule expansion, tubulointerstitial inflammatory cell infiltration, glomerular basement membrane hyperplasia and obvious interstitial fibrosis. Western blot, immunofluorescence and immunohistochemistry staining showed that the expression levels of Vimentin, PCNA and α-SMA in kidney tissue were elevated ( P<0.000 1). After PBOO, the bladder, ureter, and kidney of the mice showed obvious morphological alteration and presented reflux renal fibrosis-like damage. This can be used as an animal model to study the pathological alteration mechanism and therapeutic measures of renal fibrosis caused by bladder outlet obstruction.