Glomerular Scarring Research Articles

Endothelin Inhibitors in Chronic Kidney Disease: New Treatment Prospects.

The endothelin system is reported to play a significant role in glomerular and tubulointerstitial kidney disease. In the kidney, endothelins are produced in mesangial cells and the glomerular basement membrane by the endothelium and podocytes. The endothelin system regulates glomerular function by inducing proliferation, increasing permeability and in effect proteinuria, and stimulating inflammation, tubular fibrosis, and glomerular scarring. Endothelin A receptor antagonists have been proven to delay the progression of chronic kidney disease and play a protective role in immunoglobulin A nephropathy, focal segmental glomerulosclerosis, and diabetic nephropathy. There are several ongoing research studies with ETAR antagonists in nondiabetic nephropathy, Alport disease, vasculitis and scleroderma nephropathy, which results are promising. Some reports suggest that the endothelin system might contribute to ischemia-reperfusion injury, acute graft rejection and deterioration of graft function. Endothelin inhibition in renal transplantation and its influence on graft survival is the future direction needing further research. The most frequent side effects associated with ETAR antagonists is fluid retention. Additionally, it should be considered if selective ETAR antagonists therapy needs to be co-administered with sodium-glucose co-transporter 2 inhibitors, renin-angiotensin-aldosterone inhibitors or diuretics and which patients should be recruited to such treatment to minimize the risk of adverse outcomes.

#2305 Mice with a PAX2 missense variant are susceptible to experimental focal segmental glomerulosclerosis (FSGS) and display impaired glomerular repair

Abstract Background and Aims Focal segmental glomerulosclerosis (FSGS) is a histopathologic lesion observed in chronic kidney disease characterized by scarring of the glomerulus due to the injury and irreversible loss of highly specialized glomerular epithelial cells called podocytes. Recent studies suggest that podocytes may be regenerated by another population of glomerular epithelial cells called parietal epithelial cells (PECs), although this remains controversial. PAX2 is a transcription factor protein that regulates glomerular development and its expression persists in adult parietal epithelial cells (PECs), postulated to serve as a reservoir for podocytes in the event of injury and loss. Our team previously found that PAX2 pathogenic missense variants account for 4% of adult FSGS. In our present study, our aim is to determine whether PEC-mediated podocyte regeneration is impaired in mice with a pathogenic Pax2 missense variant (Pax2-MV). Method FSGS was induced in wildtype and Pax2-MV mice using Adriamycin. Urine samples were collected from mice every week for 28 days to measure proteinuria. At 28 days after Adriamycin injury, kidneys were collected for histopathology, transmission electron microscopy, and immunofluorescence staining for PAX2 and podocyte marker WT-1. Glomeruli from Adriamycin-injured mice were also isolated through intra-cardiac perfusion with Dynabeads and subjected to mass-spectrometry for proteomics analysis. Proteins with statistically-significant fold changes in expression levels were further analyzed by Gene Ontology to determine biological and molecular processes involved in pathogenesis. Kidney tissue from an FSGS patient with a pathogenic PAX2 missense variant was also examined by histopathology and PAX2 immunohistochemistry. Results Compared to wildtype, Pax2-MV mice were more susceptible to worsened FSGS after Adriamycin, as demonstrated by worsened glomerular scarring, increased podocyte injury and loss, and increased proteinuria, associated with increased morality in Pax2-MV mice (Fig. 1A-B). Wildtype mice showed fewer PAX2-expressing PECs after injury, accompanied by the occurrence of PAX2/WT-1 co-expressing glomerular tuft cells (Fig. 2A-B). These suggest that in Adriamycin-injured wildtype, PAX2-expressing PECs move to the glomerular tuft, where they trans-differentiate to a podocyte fate. In contrast, Pax2-MV mice showed no changes in numbers of PAX2-expressing PECs after injury, associated with fewer PAX2/WT-1 co-expressing glomerular tuft cells compared to injured wildtype (Fig. 2A-B). These suggest that in Adriamycin-injured Pax2-MV mice, PAX2-expressing PECs are unable to move to the glomerular tuft, resulting in decreased numbers of cells undergoing podocyte trans-differentiation. A subset of ectopic PAX2-expressing glomerular tuft cells after injury was increased in Pax2-MV mice (Fig. 2A), suggesting a pathologic process given the worse outcomes observed in these mice. Consistent with findings from injured Pax2-MV mice, we found that in an FSGS patient with a pathogenic PAX2 missense variant, PAX2-expressing glomerular tuft cells overlap with areas of glomerular scarring, while in a normal human kidney biopsy, PAX2 expression is only observed in PECs but not within glomerular tuft cells (Fig. 2A). Lastly, proteomics Gene Ontology analysis of isolated glomeruli demonstrated biological and molecular processes indicative of maladaptive glomerular repair in injured Pax2-MV mice, including decreased regulation of cell shape, decreased cell-cell junction assembly and decreased actin and cadherin binding compared to wildtype (Fig. 2C). Conclusion Altogether, our findings suggest that PAX2-expressing PECs migrate to the glomerular tuft to assist in podocyte regeneration in wildtype. However, this mechanism is impaired in Pax2-MV mice, leading to worsened podocyte loss and injury and further exacerbation of glomerular disease (Fig. 2C). Our findings provide a further understanding of normal and impaired regeneration mechanisms in FSGS, and our identified pathogenic mechanisms can be further harnessed for future therapeutic development.

Mice with a Pax2 missense variant display impaired glomerular repair.

PAX2 regulates kidney development, and its expression persists in parietal epithelial cells (PECs), potentially serving as a podocyte reserve. We hypothesized that mice with a Pax2 pathogenic missense variant (Pax2A220G/+) have impaired PEC-mediated podocyte regeneration. Embryonic wild-type mouse kidneys showed overlapping expression of PAX2/Wilms' tumor-1 (WT-1) until PEC and podocyte differentiation, reflecting a close lineage relationship. Embryonic and adult Pax2A220G/+ mice have reduced nephron number but demonstrated no glomerular disease under baseline conditions. Pax2A220G/+ mice compared with wild-type mice were more susceptible to glomerular disease after adriamycin (ADR)-induced podocyte injury, as demonstrated by worsened glomerular scarring, increased podocyte foot process effacement, and podocyte loss. There was a decrease in PAX2-expressing PECs in wild-type mice after adriamycin injury accompanied by the occurrence of PAX2/WT-1-coexpressing glomerular tuft cells. In contrast, Pax2A220G/+ mice showed no changes in the numbers of PAX2-expressing PECs after adriamycin injury, associated with fewer PAX2/WT-1-coexpressing glomerular tuft cells compared with injured wild-type mice. A subset of PAX2-expressing glomerular tuft cells after adriamycin injury was increased in Pax2A220G/+ mice, suggesting a pathological process given the worse outcomes observed in this group. Finally, Pax2A220G/+ mice have increased numbers of glomerular tuft cells expressing Ki-67 and cleaved caspase-3 compared with wild-type mice after adriamycin injury, consistent with maladaptive responses to podocyte loss. Collectively, our results suggest that decreased glomerular numbers in Pax2A220G/+ mice are likely compounded with the inability of their mutated PECs to regenerate podocyte loss, and together these two mechanisms drive the worsened focal segmental glomerular sclerosis phenotype in these mice.NEW & NOTEWORTHY Congenital anomalies of the kidney and urinary tract comprise some of the leading causes of kidney failure in children, but our previous study showed that one of its genetic causes, PAX2, is also associated with adult-onset focal segmental glomerular sclerosis. Using a clinically relevant model, our present study demonstrated that after podocyte injury, parietal epithelial cells expressing PAX2 are deployed into the glomerular tuft to assist in repair in wild-type mice, but this mechanism is impaired in Pax2A220G/+ mice.

Histologic and Molecular Correlates in Patients with AL Amyloidosis in Hematologic Remission but with Persistent Renal Disease

ABSTRACT: Despite the high prevalence of kidney involvement in patients with light chain (AL) amyloidosis, a significant knowledge gap exists in understanding how immunoglobulin light chains drive kidney injury. The chief reason is the lack of molecular data from high-quality kidney tissue samples from this patient population. The RAIN trial (NCT03168906) was a phase 2b randomized placebo-controlled, clinical trial that evaluated the efficacy of NEOD001, a monoclonal antibody targeting amyloid fibrils, versus placebo in patients with AL nephropathy. Before drug administration, renal biopsies were taken and analyzed. OBJECTIVE: Identifying amyloid-specific molecular signatures in kidney biopsy tissue. MATERIALS & METHODS: Renal biopsies were collected on all RAIN participants before randomization to NEOD001 or placebo. Clinical data was collected and correlated to histologic and molecular findings. Histologic assessment included a composite scarring injury score (CSIC) as determined by the degree of glomerular and tubulointerstitial scarring, and a novel amyloid score (AS), as quantified by the extent and distribution of amyloid deposition. Glomerular (G) and tubulointerstitial (TI) compartments were micro-dissected and sequenced separately. To identify enriched pathways in AL nephropathy, the expression data was compared to the glomerular and tubular expression profiles of healthy living donor (LD) from the Nephrotic Syndrome Study Network (NEPTUNE) study. Differentially expressed genes (DEGs) were determined . RESULTS: Ten patients were enrolled, randomized and treated in the RAIN trial (5 NEOD001 and five placeboes) before trial closure in April 2018. Kidney tissue was available in nine of the ten patients. Cluster analysis revealed two distinct patient clusters (G1 and G2) within the glomerular and TI compartments ( See Figure 1). Key clinical data pertaining to kidney function and biopsy morphology by compartment and cluster type were documented. Despite similar eGFR and degree of proteinuria, there was a significantly higher AS score in the TI compartment (6.5 vs. 4.5; p=0.0290) of G2 which was driven most entirely by an increase in mesangial and capillary wall amyloid deposition. Pathway Enrichment in G2 versus G1: Glomeruli showed activation of fibrotic pathways accompanied by a reduction in metabolic processes including gluconeogenesis, lipid transport and xenobiotics. There was an increase in canonical signaling of LPS/IL-1 that drives increased IL1 and TNF release from macrophages. Transcriptomic evidence of TNF activation was also noted in the TI compartment. Other enriched ingenuity canonical pathways to note in the TI included ‘coagulation system’, ‘GADD45 signaling’, ‘Wnt/Ca+ pathway’, ‘intrinsic prothrombin activation pathway’ among others. Of note, genes like BRCA and CDKN1A were enriched between G1 and G2, both of which have been associated with molecular mechanisms that underly certain neoplasms ( See Figure 2). Pathway enrichment in Amyloid kidney disease versus LD: IPA analysis identified unique activation signatures in the glomeruli and tubules. In the glomeruli, there was increased signaling of TNF, NFKB, IL33, IL-8 and IL15, consistent with an important inflammatory response. There was also an enrichment in pathways related to migration, adhesion and integrin signaling. PI3K/Akt signaling was also noted. Tubules showed increased activation of transcriptional regulators TP53, TP73 and PI3K/AKT signaling. Some gene regulators of cellular proliferation such as ABL1, BCL2 and AKT1 were found to be enriched in the amyloid group. CONCLUSIONS: While the RAIN trial closed prematurely and NEOD001 cannot be evaluated, the molecular data revealed the presence of two distinct molecular subtypes of AL nephropathy. Whether these distinct molecular subtypes reflect different stages of the disease along a continuous spectrum or are of unique prognostic entities in of themselves remains unclear. Detailed studies, of a larger cohort of patients including investigating diseased tissue at a single cell resolution can help identify the response of individual kidney cell types. Understanding the cellular responses of key cell types to injury from amyloid deposits could provide novel therapeutic targets to mitigate renal injury and improve patient outcomes. Based on this early work, targeting the PI3K/Akt pathway may be a worthy pursuit in this disease.

Podocyte injury at young age causes premature senescence and worsens glomerular aging.

As life expectancy continues to rise, age-related diseases are becoming more prevalent. For example, proteinuric glomerular diseases typified by podocyte injury have worse outcomes in the elderly compared with young patients. However, the reasons are not well understood. We hypothesized that injury to nonaged podocytes induces senescence, which in turn augments their aging processes. In primary cultured human podocytes, injury induced by a cytopathic antipodocyte antibody, adriamycin, or puromycin aminonucleoside increased the senescence-related genes CDKN2A (p16INK4a/p14ARF), CDKN2D (p19INK4d), and CDKN1A (p21). Podocyte injury in human kidney organoids was accompanied by increased expression of CDKN2A, CDKN2D, and CDKN1A. In young mice, experimental focal segmental glomerulosclerosis (FSGS) induced by adriamycin and antipodocyte antibody increased the glomerular expression of p16, p21, and senescence-associated β-galactosidase (SA-β-gal). To assess the long-term effects of early podocyte injury-induced senescence, we temporally followed young mice with experimental FSGS through adulthood (12 m of age) and middle age (18 m of age). p16 and Sudan black staining were higher at middle age in mice with earlier FSGS compared with age-matched mice that did not get FSGS when young. This was accompanied by lower podocyte density, reduced canonical podocyte protein expression, and increased glomerular scarring. These results are consistent with injury-induced senescence in young podocytes, leading to increased senescence of podocytes by middle age accompanied by lower podocyte lifespan and health span.NEW & NOTEWORTHY Glomerular function is decreased by aging. However, little is known about the molecular mechanisms involved in age-related glomerular changes and which factors could contribute to a worse glomerular aging process. Here, we reported that podocyte injury in young mice and culture podocytes induced senescence, a marker of aging, and accelerates glomerular aging when compared with healthy aging mice.

Hidden genetics behind glomerular scars: an opportunity to understand the heterogeneity of focal segmental glomerulosclerosis?

Focal segmental glomerulosclerosis (FSGS) is a complex disease which describes different kinds of kidney defects, not exclusively linked with podocyte defects. Since nephrin mutation was first described in association with early-onset nephrotic syndrome (NS), many advancements have been made in understanding genetic patterns associated with FSGS. New genetic causes of FSGS have been discovered, displaying unexpected genotypes, and recognizing possible site of damage. Many recent large-scale sequencing analyses on patients affected by idiopathic chronic kidney disease (CKD), kidney failure (KF) of unknown origin, or classified as FSGS, have revealed collagen alpha IV genes, as one of the most frequent sites of pathogenic mutations. Also, recent interest in complex and systemic lysosomal storage diseases, such as Fabry disease, has highlighted GLA mutations as possible causes of FSGS. Tubulointerstitial disease, recently classified by KDIGO based on genetic subtypes, when associated with UMOD variants, may phenotypically gain FSGS features, as well as ciliopathy genes or others, otherwise leading to completely different phenotypes, but found carrying pathogenic variants with associated FSGS phenotype. Thus, glomerulosclerosis may conceal different heterogeneous conditions. When a kidney biopsy is performed, the principal objective is to provide an accurate diagnosis. The broad spectrum of phenotypic expression and genetic complexity is demonstrating that a combined path of management needs to be applied. Genetic investigation should not be reserved only to selected cases, but rather part of medical management, integrating with clinical and renal pathology records. FSGS heterogeneity should be interpreted as an interesting opportunity to discover new pathways of CKD, requiring prompt genotype-phenotype correlation. In this review, we aim to highlight how FSGS represents a peculiar kidney condition, demanding multidisciplinary management, andin which genetic analysis may solve some otherwise unrevealed idiopathic cases. Unfortunately there is not a uniform correlation between specific mutations and FSGS morphological classes, as the same variants may be identified in familial cases or sporadic FSGS/NS or manifest a variable spectrum of the same disease. These non-specific features make diagnosis challenging. The complexity of FSGS genotypes requires new directions. Old morphological classification does not provide much information about the responsible cause of disease and misdiagnoses may expose patients to immunosuppressive therapy side effects, mistaken genetic counseling, and misguided kidney transplant programs.

Phellinus igniarius ameliorates renal aging in a rat model of focal and segmental glomerulosclerosis

Objective: To comparatively investigate the ameliorative effect of Phellinus igniarius (P. igniarius) on renal aging in a rat model of focal and segmental glomerulosclerosis (FSGS). Methods: The FSGS model was established in rats by uninephrectomy combined with tail vein injection of doxorubicin. The FSGS rats were randomly divided into the model group, the P. igniarius decoction group, the P. igniarius polysaccharides group, and the P. igniarius polyphenols group. Molecular indicators of cell senescence, renal function indexes, and podocyte injury markers were tested after ten weeks of intragastric administration. Besides, the pathological renal lesions and the ultrastructural changes were observed. Results: FSGS developed in the model group within ten weeks and showed segmental glomerular scarring and renal aging. Following the 10-week intervention, 24 h proteinuria, serum creatinine, blood urea nitrogen, P16INK4α, thrombospondin-1, and transforming growth factor-β1 were decreased in each treatment group, whereas albumin, erythropoietin, nephrin, and podocin were increased; the pathological renal injury was alleviated, and the number of senescent cells was reduced, especially in rats treated with P. igniarius decoction. Conclusions: P. igniarius ameliorates renal aging and renal injury in the FSGS rat model. Compared with the effective constituents (polysaccharides and polyphenols), P. igniarius decoction has a better curative effect, which is expected to provide a new therapeutic idea for FSGS.

High fat diet is protective against kidney injury in hypertensive-diabetic mice, but leads to liver injury.

Chronic kidney disease (CKD) is a worldwide health burden with increases risk of end-stage renal function if left untreated. CKD induced in the context of metabolic syndrome (MS) increases risks of hypertension, hyperglycemia, excess body fat and dyslipidemia. To test if combining a high-fat diet (HFD) regimen onto the hypertensive/ diabetic phenotype would mimic features of MS induced-CKD in mice, hyperglycemia was induced in genetically hypertensive mice (Lin), followed by HFD regimen. For that, 8-week-old male were subjected to streptozotocin (STZ) intraperitoneal (i.p.) injections (50 mg/kg, 5 days consecutive). LinSTZ were fed a 60% kCal HFD for 8 weeks. Lin mice treated with STZ developed polydipsia, became hypertensive and hyperglycemic. HFD induced weight gain, protected against glomerular hypertrophy, scarring, and albuminuria at endpoint compared to regular diet fed LinSTZ. On the other hand, HFD induced steatosis, liver fibrosis, inflammation, and increase in AST/ALT ratio, characteristics of non-alcoholic liver disease. Taken together, our results show that LinSTZ mice fed a HFD did not lead to a more robust model of MS-induced CKD, protected against kidney injury, but inducing liver damage. More studies are necessary to understand the kidney protective mechanisms of HFD when superimposed with hypertension and type 1 diabetes.

Gene Expression Sets and Renal Profiling from the Rain (Renal AL Amyloid Involvement and NEOD00) Trial

INTRODUCTION: Despite the high prevalence of kidney involvement in patients with light chain (AL) amyloidosis, a significant knowledge gap exists in understanding how immunoglobulin light chains drive kidney injury. The chief reason is the lack of molecular data from high-quality kidney tissue samples from this patient population. The RAIN trial (NCT03168906) was a phase 2B randomized placebo-controlled, open-label clinical trial that evaluated the efficacy of NEOD001, a monoclonal antibody targeting amyloid fibrils, versus placebo in patients with AL nephropathy. OBJECTIVES: Identifying amyloid-specific molecular signatures in kidney biopsy tissue obtained from AL patients with nephropathy.MATERIALS& METHODS: Renal biopsies were collected on all RAIN participants before randomization to NEOD001 or placebo. Trial clinical data was incorporated to link tissue, molecular, and kidney function data. The histologic assessment included a composite scarring injury score (CSIC) as determined by the degree of glomerular and tubulointerstitial scarring, and a novel amyloid score (AS), as quantified by the extent and distribution of amyloid deposition. Glomerular (G) and tubulointerstitial compartments were micro-dissected and sequenced separately. To identify enriched pathways in AL nephropathy, the expression data was compared to the glomerular and tubular expression profiles of healthy living donor (LD) from the Nephrotic Syndrome Study Network (NEPTUNE) study. Differentially expressed genes (DEGs) were determined with the R limma package based on the voom transformed log2 count data with an FDR of <=0.05. The AL and NEPTUNE datasets were merged and batch-corrected using ComBat. RESULTS: Ten patients were enrolled and randomized in the RAIN trial (5 NEOD001 and five placeboes) before trial closure in April 2018. Kidney tissue was available in nine of the ten patients. Cluster analysis revealed two distinct patient clusters (G1 and G2) within the glomerular and tubulointerstitial compartments. Key data of kidney function and biopsy morphology by compartment and cluster type are shown in Table 1. Despite similar eGFR, there was a higher AS and a non-significant increase in 24-hour urine protein in the glomeruli of individuals in the G2 patient cluster. Subcomponent analysis of AS revealed that the increase in amyloid score in G2 was driven almost entirely by an increase in mesangial and capillary wall amyloid deposition. Pathway enrichment in Amyloid kidney disease versus LD: IPA analysis identified unique activation signatures in the glomeruli and tubules. In the glomeruli of biopsies from the G2 cluster, there was evidence of increased TNF, IFNG, IL33 and VEGFA cytokine activity and a predicted increase in canonical PI3K/Akt signaling and innate immune responses. Tubules showed increased activation of transcriptional regulators TP53, TP73, increased cytokine signaling of IFNG, TGFb1 and VEGF. There was also a predicted activation of major canonical pathways including B cell receptor signaling, PI3K/AKT and Ephrin receptor signaling (Fig 1). Pathway Enrichment in G2 versus G1: Glomeruli showed activation of fibrotic pathways accompanied by a reduction in metabolic processes including gluconeogenesis, lipid transport and xenobiotics. There was an increase in canonical signaling of LPS/IL-1 mediated inhibition of RXR function that drives increased IL1 and TNF release from macrophages. Transcriptomic evidence of TNF activation was also noted in the TI. CONCLUSIONS: The RAIN trial closed prematurely, and thus no conclusions can be reached about the efficacy of NEOD001 in patients with AL nephropathy. However, these limited data revealed the presence of two distinct molecular subtypes of AL nephropathy. Whether these distinct molecular subtypes reflect different stages of the disease along a continuous spectrum or are of unique prognostic entities in themselves remains unclear. Detailed studies, of a larger cohort of patients including investigating diseased tissue at a single cell resolution can help identify the response of individual kidney cell types. Understanding the cellular responses of key cell types to injury from amyloid deposits could provide novel therapeutic targets to mitigate renal injury and improve patient outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

An adapted passive model of anti-MPO dependent crescentic glomerulonephritis reveals matrix dysregulation and is amenable to modulation by CXCR4 inhibition

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are severe inflammatory disorders that often involve focal necrotizing glomerulonephritis (FNGN) and consequent glomerular scarring, interstitial fibrosis, and chronic kidney disease. Robust murine models of scarring in FNGN that may help to further our understanding of deleterious processes are still lacking. Here, we present a murine model of severe FNGN based on combined administration of antibodies against the glomerular basement membrane (GBM) and myeloperoxidase (MPO), and bacterial lipopolysaccharides (LPS), that recapitulates acute injury and was adapted to investigate subsequent glomerular and interstitial scarring. Hematuria without involvement of other organs occurs consistently and rapidly, glomerular necrosis and crescent formation are evident at 12 days, and consequent glomerular and interstitial scarring at 29 days after initial treatment. Using mass-spectrometric proteome analysis, we provide a detailed overview of matrisomal and cellular changes in our model. We observed increased expression of the matrisome including collagens, fibronectin, tenascin-C, in accordance with human AAV as deduced from analysis of gene expression microarrays and tissue staining. Moreover, we observed tissue infiltration by neutrophils, macrophages, T cells and myofibroblasts upon injury. Experimental inhibition of CXCR4 using AMD3100 led to a sustained histological presence of fibrin extravasate, reduced chemokine expression and leukocyte activation, but did not markedly affect ECM composition. Altogether, we demonstrate an adapted FNGN model that enables the study of matrisomal changes both in disease and upon intervention, as exemplified via CXCR4 inhibition.

Characterisation of an enhanced preclinical model of experimental MPO-ANCA autoimmune vasculitis.

Experimental autoimmune vasculitis (EAV) is a model of antineutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV) induced by immunisation of susceptible rat strains with myeloperoxidase (MPO). Animals develop circulating MPO-ANCA, pulmonary haemorrhage, and glomerulonephritis, although renal injury is mild and recovers spontaneously without treatment. In this study we aimed to augment the severity of glomerulonephritis. Following induction of EAV on day 0, a sub-nephritogenic dose of nephrotoxic serum (NTS) containing heterologous antibodies to glomerular basement membrane was administered on day 14. This resulted in a significant increase in disease severity at day 28 compared to MPO immunisation alone - with more urinary abnormalities, infiltrating glomerular leucocytes, and crescent formation that progressed to glomerular and tubulointerstitial scarring by day 56, recapitulating important features of human disease. Importantly, the glomerulonephritis remained pauci-immune, and was strictly dependent on the presence of autoimmunity to MPO, as there was no evidence of renal disease following administration of sub-nephritogenic NTS alone or after immunisation with a control protein in place of MPO. Detailed phenotyping of glomerular leucocytes identified an early infiltrate of non-classical monocytes following NTS administration that, in the presence of autoimmunity to MPO, may initiate the subsequent influx of classical monocytes which augment glomerular injury. We also showed that this model can be used to test novel therapeutics by using a small molecule kinase inhibitor (fostamatinib) that rapidly attenuated both glomerular and pulmonary injury over a 4-day treatment period. We believe that this enhanced model of MPO-AAV will prove useful for the study of glomerular leucocyte behaviour and novel therapeutics in AAV in the future. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Characterization of the Peritumoral Atrophic Band and Nonneoplastic Renal Parenchyma in Radical Nephrectomy Specimens.

Pathologic evaluation of nonneoplastic renal parenchyma in nephrectomy specimens is important for identifying chronic kidney disease and diabetic nephropathy, but increasing utilization of partial nephrectomies has led to less-sampled nonneoplastic parenchyma. The sampled tissue is often composed predominantly of the peritumoral atrophic band (PAB) directly adjacent to the tumor. We sought to determine the characteristics of the PAB and whether it could be used to reliably assess kidney pathology, including diabetic nephropathy. We investigated 59 radical nephrectomies to determine the PAB characteristics, whether the PAB is representative of distant nonneoplastic parenchyma, and if diabetic nephropathy could be reliably detected in the PAB. Mesangial sclerosis was detected within the PAB in 100% of cases with mesangial sclerosis in the distant parenchyma. Eighty percent had a history of diabetes. The PAB exhibited increased glomerular sclerosis (51% vs 13%, P < .001) and interstitial fibrosis and tubular atrophy (83% vs 13%, P < .001) compared with distant parenchyma. Diabetic nephropathy can be reliably detected in the PAB, which is important in partial nephrectomies or renal mass biopsies without ample distant renal parenchyma. The degree of glomerular and tubulointerstitial scarring within the PAB does not reflect the overall degree of chronic kidney disease.

The Exostosin Immunohistochemical Status Differentiates Lupus Membranous Nephropathy Subsets With Different Outcomes

The Exostosin Immunohistochemical Status Differentiates Lupus Membranous Nephropathy Subsets With Different Outcomes

More than ancillary records: clinical implications of renal pathology examination in tumor nephrectomy specimens

BackgroundNephrectomy is the management of choice for the treatment of renal tumors. Surgical pathologists primarily focus on tumor diagnosis and investigations relating to prognosis or therapy. Pathological changes in non-neoplastic tissue may, however, be relevant for further management and should be thoroughly assessed.MethodsHere, we examined the non-neoplastic renal parenchyma in 206 tumor nephrectomy specimens for the presence of glomerular, tubulo-interstitial, or vascular lesions, and correlated them with clinical parameters and outcome of renal function.ResultsWe analyzed 188 malignant and 18 benign or pseudo-tumorous lesions. The most common tumor type was clear cell renal cell carcinoma (CCRCC, n = 106) followed by papillary or urothelial carcinomas (n = 25). Renal pathology examination revealed the presence of kidney disease in 39 cases (18.9%). Glomerulonephritis was found in 15 cases (7.3%), and the most frequent was IgA nephropathy (n = 6; 2.9%). Vasculitis was found in two cases (0.9%). In 15 cases we found tubulo-interstitial nephritis, and in 9 severe diabetic or hypertensive nephropathy. Partial nephrectomy was not linked to better eGFR at follow-up. Age, vascular nephropathy, glomerular scarring and interstitial fibrosis were the leading independent negative factors influencing eGFR at time of surgery, whereas proteinuria was associated with reduced eGFR at 1 year.ConclusionOur large study population indicates a high incidence of renal diseases potentially relevant for the postoperative management of patients with renal neoplasia. Consistent and systematic reporting of non-neoplastic renal pathology in tumor nephrectomy specimens should therefore be mandatory.

PODO: Trial Design: Phase 2 Study of PF-06730512 in Focal Segmental Glomerulosclerosis

IntroductionFocal segmental glomerulosclerosis (FSGS) is characterized by proteinuria and a histologic pattern of glomerular lesions of diverse etiology that share features including glomerular scarring and podocyte foot process effacement. Roundabout guidance receptor 2 (ROBO2)/slit guidance ligand 2 (SLIT2) signaling destabilizes the slit diaphragm and reduces podocyte adhesion to the glomerular basement membrane (GBM). Preclinical studies suggest that inhibition of glomerular ROBO2/SLIT2 signaling can stabilize podocyte adhesion and reduce proteinuria. This clinical trial evaluates the preliminary efficacy and safety of ROBO2/SLIT2 inhibition with the ROBO2 fusion protein PF-06730512 in patients with FSGS.MethodsThe Study to Evaluate PF-06730512 in Adults With FSGS (PODO; ClinicalTrials.gov identifier NCT03448692), an open-label, phase 2a, multicenter trial in adults with FSGS, will enroll patients into 2 cohorts (n = 22 per cohort) to receive either high- or low-dose PF-06730512 (intravenous) every 2 weeks for 12 weeks. Key inclusion criteria include a confirmed biopsy diagnosis of FSGS, an estimated glomerular filtration rate (eGFR) ≥45 ml/min/1.73 m2 based on the Chronic Kidney Disease Epidemiology Collaboration formula (30–45 with a recent biopsy), and urinary protein-to-creatinine ratio (UPCR) >1.5 g/g. Key exclusion criteria include collapsing FSGS, serious/active infection, ≥50% tubulointerstitial fibrosis on biopsy, and organ transplantation. The primary endpoint is change from baseline to week 13 in UPCR; secondary endpoints include safety, changes in eGFR, and PF-06730512 serum concentration.ResultsThis ongoing trial will report the efficacy, safety, pharmacokinetics, and biomarker results of PF-06730512 for patients with FSGS.ConclusionFindings from this proof-of-concept study may support further development and evaluation of PF-06730512 to treat FSGS and warrant assessment in phase 3 clinical trials.

POS-369 CHARACTERIZATION OF A NOVEL MOUSE MODEL OF METABOLIC SYNDROME INDUCED-CHRONIC KIDNEY DISEASE

Chronic kidney disease (CKD) induced in the context of metabolic syndrome (MS) involves underlying hypertension, hyperglycemia, excess body fat and dyslipidemia. A previously generated renin-dependent hypertensive/type-1 diabetic mouse model develops several signs associated human DKD, however the extent and impact of dyslipidemia in this model remains unknown. We hypothesized that combining a high-fat diet (HFD) regimen onto the hypertensive/ diabetic phenotype would mimic features of MS induced-CKD in mice. To this end, 8-week old male genetically hypertensive mice (Lin+) were subjected to streptozotocin (STZ) injections (i.p., 50 mg/kg, 5 days consecutive) to induce hyperglycemia. Four-week later hypertensive/ diabetic mice (LinSTZ) were fed a HFD for 8 weeks. Blood pressure, body weight and blood glucose (BG) were measured throughout the study, while urine albumin levels and histological assessment were performed at endpoint. A HFD-regimen led to significant weight gain and increased circulating triglycerides in WT and to lesser extent LinSTZ mice. Throughout the study, HFD-feeding seemed to reduce systolic blood pressure in LinSTZ mice, while BG remained elevated. Surprisingly, HFD-fed LinSTZ mice developed less glomerular hypertrophy, scarring and albuminuria at endpoint than regular-diet fed littermates. In contrast, histological assessment of liver injury revealed that both WT+HFD and LinSTZ mice developed steatosis (fat accumulation) lesions, while HFD-feeding in LinSTZ mice led to pronounced hepatocyte ballooning, liver inflammation and fibrosis. Moreover, circulating albumin levels, as a surrogate for liver function, were significantly albeit similarly reduced in both vehicle and HFD-fed LinSTZ mice. Taken together, our results show that HFD-feeding in LinSTZ did not lead to a more robust model of MS-induced CKD. In fact, several indices of renal injury were reduced by feeding LinSTZ mice a HFD. The combination of renin-driven hypertension and STZ-induced type-1 diabetes did however accelerate the development of liver injury in the context of MS. Subsequent studies will be undertaken to determine whether feeding LinSTZ a HFD may represent a novel model of accelerated non-alcoholic fatty liver disease.

CALINCA-A Novel Pipeline for the Identification of lncRNAs in Podocyte Disease.

Diseases of the renal filtration unit—the glomerulus—are the most common cause of chronic kidney disease. Podocytes are the pivotal cell type for the function of this filter and focal-segmental glomerulosclerosis (FSGS) is a classic example of a podocytopathy leading to proteinuria and glomerular scarring. Currently, no targeted treatment of FSGS is available. This lack of therapeutic strategies is explained by a limited understanding of the defects in podocyte cell biology leading to FSGS. To date, most studies in the field have focused on protein-coding genes and their gene products. However, more than 80% of all transcripts produced by mammalian cells are actually non-coding. Here, long non-coding RNAs (lncRNAs) are a relatively novel class of transcripts and have not been systematically studied in FSGS to date. The appropriate tools to facilitate lncRNA research for the renal scientific community are urgently required due to a row of challenges compared to classical analysis pipelines optimized for coding RNA expression analysis. Here, we present the bioinformatic pipeline CALINCA as a solution for this problem. CALINCA automatically analyzes datasets from murine FSGS models and quantifies both annotated and de novo assembled lncRNAs. In addition, the tool provides in-depth information on podocyte specificity of these lncRNAs, as well as evolutionary conservation and expression in human datasets making this pipeline a crucial basis to lncRNA studies in FSGS.

Endocan levels in children with renal hypodysplasia.

Severe reduction in nephron numbers that are characteristic of renal hypodysplasia (RHD) are one of the cause of childhood chronic kidney disease (CKD). Glomerular hyperfiltration, glomerular hypertrophy, progressive glomerular scarring, and interstitial fibrosis due to reduced nephron number are risk factors for CKD. In recent years, studies on specific markers for early diagnosis of renal failure and mortality have been carried out. The objectives of this study were to identify serum and urinary endocan levels that are expressed in glomerular endothelial cells and tubular epithelial cells in RHD. 29 children with RHD were compared to 26 healthy controls in terms of serum and urinary endocan levels. The mean serum endocan level in the RHD group and the control group was 700.72 ± 323.19 and 426.86 ± 233.14 pg/mL, respectively. The mean serum endocan level was significantly higher (p = 0.003) in the RHD group. The mean urinary endocan level in the RHD group was 63.62 ± 92.46 pg/mL, and in the control group it was 80.26 ± 142.49 pg/mL. The mean urinary endocan level did not change between groups (p = 0.95). There was also a significant correlation between serum endocan level and uric acid level in the study group (r = 0.325, p = 0.028). To our knowledge, this was the first study that evaluated serum and urinary endocan levels in children with RHD. Although serum endocan level was found to be significantly higher in patients with RHD, further studies are needed to validate whether endocan could serve as a marker of poor renal prognosis in RHD.

Calpastatin prevents Angiotensin II–mediated podocyte injury through maintenance of autophagy

The strong predictive value of proteinuria in chronic glomerulopathies is firmly established as well as the pathogenic role of angiotensin II promoting progression of glomerular disease with an altered glomerular filtration barrier, podocyte injury and scarring of glomeruli. Here we found that chronic angiotensin II-induced hypertension inhibited autophagy flux in mouse glomeruli. Deletion of Atg5 (a gene encoding a protein involved autophagy) specifically in the podocyte resulted in accelerated angiotensin II-induced podocytopathy, accentuated albuminuria and glomerulosclerosis. This indicates that autophagy is a key protective mechanism in the podocyte in this condition. Angiotensin-II induced calpain activity in podocytes inhibits autophagy flux. Podocytes from mice with transgenic expression of the endogenous calpain inhibitor calpastatin displayed higher podocyte autophagy at baseline that was resistant to angiotensin II-dependent inhibition. Also, sustained autophagy with calpastatin limited podocyte damage and albuminuria. These findings suggest that hypertension has pathogenic effects on the glomerular structure and function, in part through activation of calpains leading to blockade of podocyte autophagy. These findings uncover an original mechanism whereby angiotensin II-mediated hypertension inhibits autophagy via calcium-induced recruitment of calpain with pathogenic consequences in case of imbalance by calpastatin activity. Thus, preventing a calpain-mediated decrease in autophagy may be a promising new therapeutic strategy for nephropathies associated with high renin-angiotensin system activity.

Histopathological Findings Predict Renal Recovery in Severe ANCA-Associated Vasculitis Requiring Intensive Care Treatment.

Renal involvement is a common and severe complication of AAV as it can cause ESRD. Histopathological subgrouping and ARRS are helpful to predict long-term ESRD in patients with AAV. Because a subgroup of critically ill patients with severe AAV present with deterioration of kidney function requiring RRT at admission, we here aimed to evaluate histopathological findings and predictive value of Berden's histopathological subgrouping and ARRS for severity of AKI and requirement of RRT during the short-term clinical course in critically ill patients requiring intensive care treatment and predictors for short-term renal recovery in patients requiring RRT. A subgroup of 15/46 (32. 6%) AAV patients with biopsy-proven AAV required RRT during the short-term course of disease, associated with requirement of critical care treatment. While histopathological subgrouping and ARRS were associated with requirement of acute RRT, presence of global glomerular scarring was the strongest predictor of failure to recover from RRT after initiation of remission induction therapy. This new aspect requires further investigation in a prospective controlled setting for therapeutic decision making especially in this subgroup.