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Kidney organoids derived from pluripotent stem cells and epithelial organoids derived from adult tissue (tubuloids) have been used to study various kidney disorders with a strong genetic component, such as polycystic kidney disease, Wilms tumor, and congenital nephrotic syndrome. However, complex disorders without clear genetic associations, such as acute kidney injury and many forms of chronic kidney disease, are only just beginning to be investigated using these in vitro approaches. Although organoids are a reductionist model, they contain clinically relevant cell populations that may help to elucidate human-specific pathogenic mechanisms. Thus, organoids may complement animal disease models to accelerate the translation of laboratory proof-of-concept research into clinical practice. This review discusses whether kidney organoids and tubuloids are suitable models for the study of complex human kidney disease and highlights their advantages and limitations compared with monolayer cell culture and animal models., (Copyright © 2022 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Patients with diabetes are at an increased risk for acute kidney injury (AKI) after renal ischemia/reperfusion injury (IRI). However, there is a lack preclinical models of IRI in established diabetes. The current study characterized renal IRI in mice with established diabetes and investigated potential therapies. Diabetes was induced in C57BL/6J mice by low-dose streptozotocin injection. After 7 weeks of sustained diabetes, mice underwent 13 minutes of bilateral renal ischemia and were euthanized after 24 hours of reperfusion. Age-matched, nondiabetic controls underwent the same surgical procedure. Renal IRI induced two- and sevenfold increases in plasma creatinine level in nondiabetic and diabetic mice, respectively (P < 0.001). Kidney damage, as indicated by histologic damage, tubular cell death, tubular damage markers, and inflammation, was more severe in the diabetic IRI group. The diabetic IRI group showed greater accumulation of spleen tyrosine kinase (Syk)-expressing cells, and increased c-Jun N-terminal kinase (Jnk) signaling in tubules compared to nondiabetic IRI. Prophylactic treatment with a Jnk or Syk inhibitor substantially reduced the severity of AKI in the diabetic IRI model, with differential effects on neutrophil infiltration and Jnk activation. In conclusion, established diabetes predisposed mice to renal IRI-induced AKI. Two distinct proinflammatory pathways, JNK and SYK, were identified as potential therapeutic targets for anticipated AKI in patients with diabetes., (Copyright © 2022 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Activation of the JUN amino-terminal kinase (JNK) pathway is prominent in most forms of acute and progressive tubulointerstitial damage, including acute renal ischemia/reperfusion injury (IRI). Two forms of JNK, JNK1 and JNK2, are expressed in the kidney. Systemic administration of pan-JNK inhibitors suppresses renal IRI; however, the contribution of JNK1 versus JNK2, and the specific role of JNK activation in the proximal tubule in IRI, remains unknown. These questions were addressed in rat and mouse models of acute bilateral renal IRI. Administration of the JNK inhibitor, CC-930, substantially reduced the severity of renal failure, tubular damage, and inflammation at 24 hours in a rat IRI model. Additionally, Jnk1 -/- mice, but not Jnk2 -/- mice, were shown to be significantly protected against acute renal failure, tubular damage, and inflammation in the IRI model. Furthermore, mice with conditional Jnk1 deletion in the proximal tubule also showed considerable protection from IRI-induced renal failure, tubular damage, and inflammation. Finally, primary cultures of Jnk1 -/- , but not Jnk2 -/- , tubular epithelial cells were protected from oxidant-induced cell death, in association with preventing phosphorylation of proteins (receptor interacting serine/threonine kinase 3 and mixed lineage kinase domain-like pseudokinase) in the necroptosis pathway. In conclusion, JNK1, but not JNK2, plays a specific role in IRI-induced cell death in the proximal tubule, leading to acute renal failure., (Copyright © 2021 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Ischemia/reperfusion (I/R) injury is an important cause of acute and chronic renal failure. Neutrophils and macrophages, by integrin-based recruitment, play a key role in renal I/R injury. Integrin-based activation of spleen tyrosine kinase (Syk) contributes to myeloid cell adhesion to activated endothelial cells in vitro; however, whether Syk is required for myeloid cell recruitment and tubular damage in I/R injury is unknown. Therefore, we investigated the function of Syk in mouse I/R injury using two different approaches. C57Bl/6J mice underwent bilateral warm ischemia and were sacrificed after 30 minutes or 24 hours of reperfusion. Mice were treated with the Syk inhibitor CC0417, or vehicle, beginning 1 hour before surgery. Syk was expressed by infiltrating neutrophils, macrophages, and platelets in vehicle-treated I/R injury which exhibited severe renal failure and tubular damage at 24 hours. CC0417 treatment markedly reduced neutrophil, macrophage, and platelet accumulation with improved renal function and reduced tubular damage. Next, we compared mice with conditional Syk gene deletion in myeloid cells (Syk(My)) versus Syk(f/f) littermate controls in a 24-hour study. Syk(My) mice also showed a marked reduction in neutrophil and macrophage infiltration with significant protection from I/R-induced acute renal failure and tubular damage. These studies define a pathologic role for myeloid Syk signaling in renal I/R injury and identify Syk as a potential therapeutic target in this condition., (Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Transforming growth factor-β (TGF-β) promotes tissue fibrosis via receptor-mediated phosphorylation of the receptor-activated Smad2/3, together with Smad4. Of these, Smad3 plays a major profibrotic role in mouse models of tissue fibrosis. Transcriptional activity of the Smad3 protein is regulated by phosphorylation of residues in the C-terminal domain and the linker region. Herein, we examined the role of a novel phosphorylation site within the MH2 domain (T388) in the regulation of Smad3 activity. Confocal microscopy using an Smad3 phosphorylated T388-specific antibody identified phosphorylation of Smad3 T388 in myofibroblasts and tubular epithelial cells in human focal and segmental glomerulosclerosis and mouse models of unilateral ureteric obstruction and diabetic nephropathy, whereas phosphorylated T388 was largely absent in normal kidney. In vitro, TGF-β1 induced phosphorylation of Smad3 T388 in a biphasic pattern. A point mutation of T388/V in an Smad3 construct demonstrated that phosphorylation of T388 promotes Smad3 binding to Smad4 and CDK8, but was not necessary for nuclear translocation. Furthermore, T388 phosphorylation was required for TGF-β-induced collagen I gene promoter activity and extracellular matrix production in cultured fibroblasts. In conclusion, our study identifies phosphorylation of T388 in the Smad3 MH2 domain as an important mechanism that regulates the profibrotic TGF-β/Smad3 signaling pathway, which has direct relevance to human and experimental fibrotic kidney disease., (Copyright © 2014. Published by Elsevier Inc.)

Transforming growth factor-beta1 (TGF-beta1) promotes tissue fibrosis through the Smad3 signaling pathway. While phosphorylation is known to regulate Smad3 function, recent in vitro studies have suggested that acetylation may also regulate Smad3 function. This study investigated Smad3 acetylation in renal fibrosis. TGF-beta1 stimulation of renal fibroblasts and tubular epithelial cells induced Smad3 acetylation and phosphorylation. Resveratrol, an activator of the Nicotinamide adenine dinucleotide (NAD) dependent protein deacetylase SIRT1, reversed acetylation but not phosphorylation of Smad3 and inhibited TGF-beta1-induced up-regulation of collagen IV and fibronectin mRNA levels. Knockdown of SIRT1 expression abolished the inhibitory effect of resveratrol, and co-immunoprecipitation studies provide direct evidence of an interaction between acetylated Smad3 and SIRT1. The role of Smad3 acetylation in renal fibrosis was then examined in the unilateral ureteric obstruction (UUO) model. Immunoprecipitation studies showed acetylation and phosphorylation of Smad3 by day 2 UUO, which was sustained to day 7 in association with development of interstitial fibrosis. Resveratrol inhibited acetylation but not phosphorylation of Smad3 at day 2 UUO, and resveratrol treatment inhibited interstitial fibrosis at day 7 UUO. In conclusion, these studies support a pathological role for Smad3 acetylation in renal fibrosis and suggest that deacetylation of Smad3 may be a novel therapeutic target for fibrotic disease.

The increased fractional clearance of albumin in nephrotic states has long been attributed to glomerular permselectivity dysfunction. Using radiolabeled rat serum albumin, transferrin, IgG, and polydisperse Ficoll, this study investigated the changes in their in vivo fractional clearance in puromycin aminonucleoside nephrosis and anti-glomerular basement membrane glomerulonephritis. In control rats the lack of charge selectivity was confirmed by the demonstration that carboxymethyl Ficoll (valence approximately -39) had the same fractional clearance as uncharged Ficoll. Both diseases exhibited similar effects on fractional clearance measurements suggesting an underlying common mechanism. In disease, there was good agreement between the fractional clearance of proteins determined by radioactivity as compared to those determined by radioimmunoassay. A small increase in the fractional clearance for IgG was evident in disease as compared to controls, which mirrored the change in the equivalent size Ficoll, suggesting that the increase is because of the development of a small proportion of large pores in the glomerular capillary wall. There was no increase, however, in the fractional clearance of Ficoll of equivalent size to albumin in either disease, yet the fractional clearance of the albumin increased by 12 to 14 times as determined by radioactivity and 4500 to 6600 times as determined by radioimmunoassay. This study demonstrates that glomerulonephritis is not a disease associated with changes in glomerular permeability to albumin but is because of alterations in albumin processing by cells distal to the glomerular basement membrane. It is also apparent that approaches to glomerular pathology and proteinuria as risk factors in renal disease must be reassessed.

Osteopontin (OPN) is a macrophage chemotactic and adhesion molecule that acts to promote macrophage infiltration in rat anti-glomerular basement membrane (GBM) glomerulonephritis. The present study investigated the role of interleukin-1 (IL-1) in the up-regulation of renal OPN expression in this disease model. Accelerated anti-GBM glomerulonephritis was induced in groups of six rats. Animals were treated by a constant infusion of the IL-1 receptor antagonist or saline (control) over days -1 to 14 (induction phase) or days 7 to 21 (established disease). In normal rat kidney, OPN was expressed in a few tubules (<5%) and absent from glomeruli. During the development of rat anti-GBM disease (days 7 to 21), there was substantial up-regulation of OPN mRNA and protein expression in glomeruli (>5 cells per glomerular cross-section) and tubular epithelial cells (50-75% OPN-positive). Up-regulation of OPN expression was associated with macrophage accumulation within the kidney, severe proteinuria, loss of renal function, and severe histological damage including glomerular crescentic formation and tubulointerstitial fibrosis. In contrast, IL-1 receptor antagonist treatment of either the induction phase of disease or established disease significantly reduced OPN mRNA and protein expression in glomeruli (/75-85%, P < 0.001) and tubules (/45-60%, P < 0.001). The reduction in OPN expression was associated with significant inhibition of macrophage accumulation and progressive renal injury. In vitro, the addition of IL-1 to the normal rat tubular epithelial cell line NRK52E up-regulated OPN mRNA and protein levels, an effect that was dose-dependent and inhibited by the addition of IL-1 receptor antagonist, thus demonstrating that IL-1 can act directly to up-regulate renal OPN expression. In conclusion, this study provides in vivo and in vitro evidence that IL-1 up-regulates OPN expression in experimental kidney disease and support for the argument that inhibition of OPN expression is one mechanism by which IL-1 receptor antagonist treatment suppresses macrophage-mediated renal injury.

The fate of macrophages at the site of inflammation is unknown. We investigated this question in a macrophage-mediated model of crescentic glomerulonephritis in which macrophage accumulation is relatively stable despite the presence of high levels of local macrophage proliferation. Accelerated anti-glomerular basement membrane glomerulonephritis was induced in groups of six rats that were killed on day 1, 7, 14, or 21. Macrophage apoptosis was demonstrated in kidney sections by three methods: in situ terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) combined with ED1 antibody immunostaining of macrophages, ED1 immunostaining combined with classical nuclear morphology, and electron microscopy. Substantial macrophage apoptosis became evident on day 14 of the disease, following the appearance of high levels of macrophage proliferation. The parallel relationship between proliferation and apoptosis is the likely explanation for the stabilization of macrophage numbers within the inflamed kidney. A striking feature was that macrophage proliferation and apoptosis was largely restricted to areas of focal damage, such as in the development of glomerular crescents. Increasing levels of macrophage proliferation and apoptosis were evident as crescents developed from a cellular to a fibrocellular phenotype, with a dramatic reduction in both of these processes in the progression to a fibrotic phenotype, suggesting an important role for macrophage apoptosis in the resolution of fibrocellular crescents to an acellular fibrotic structure. In conclusion, this study has identified apoptosis as an important mechanism counterbalancing local proliferation in the regulation of macrophage accumulation at sites of inflammation. Indeed, apoptosis may be a central regulator of the progression and resolution of macrophage-mediated tissue injury.

We examined the functional role of interleukin (IL)-1 in mesangial cell proliferation during rat anti-Thy-1 nephritis by blocking its action with IL-1 receptor antagonist (IL-1ra). Anti-Thy-1 nephritis was induced by intravenous injection of 5 mg/kg OX-7 IgG (day 0) into inbred Wistar rats. Groups of animals (n = 9) were implanted with a micro-osmotic pump on day -1, which delivered 25 micrograms/hour human recombinant IL-1ra or saline continuously until the rats were killed at day 6, the peak of mesangial cell proliferation. Immunostaining showed that IL-1 was expressed by mesangial cells during disease. IL-1ra treatment did not affect the mild, but significant, proteinuria seen after OX-7 injection. Compared with saline treatment, IL-1ra treatment reduced mesangial cell proliferation (decreases 24% P < 0.05), glomerular hypercellularity (decreases 29%; P < 0.05), and glomerular macrophage accumulation (decreases 20%; P < 0.05). However, IL-1ra treatment had no effect on glomerular IL-1 beta mRNA expression and caused only a small reduction in the high levels of glomerular expression of platelet-derived growth factor-beta protein (decreases 6%; P < 0.05). IL-1ra caused a modest reduction in the marked up-regulation of glomerular transforming growth factor-beta 1 mRNA expression on day 6 (decreases 26%; P < 0.05), although urinary excretion of this factor was unaffected. Interestingly, IL-1ra treatment had relatively little effect upon glomerular deposition of laminin, fibronectin, and collagen type IV seen in this acute disease. In conclusion, this study has 1) demonstrated that IL-1 is expressed by mesangial cells in vivo, 2) demonstrated that IL-1 is a mesangial cell growth factor in experimental mesangioproliferative nephritis, and 3) suggests that IL-1 has little or no fibrogenic activity in mesangial matrix deposition.

Macrophage migration inhibitory factor (MIF), a key mediator of the delayed-type hypersensitivity response, was originally thought to be produced by activated T cells. However, recent studies have found that MIF is produced in many cell types including monocytes/macrophages and anterior pituitary cells. The current study has examined MIF expression in normal and diseased kidney using in situ hybridization, immunohistochemistry, and Northern blotting. MIF mRNA and protein are constitutively expressed in normal kidney, being largely restricted to tubular epithelial cells and some glomerular visceral and parietal epithelial cells. During the development of rat anti-glomerular basement membrane glomerulonephritis, a model of macrophage-mediated renal injury, there was marked de novo expression of MIF by intrinsic kidney cells including endothelium and glomerular and tubular epithelial cells. Up-regulation of MIF expression correlated with macrophage accumulation within the glomerulus (P < 0.001) and tubulointerstitium (P < 0.001). Of significance, the accumulation of macrophages was exclusively localized to areas of strong MIF expression, contributing to focal glomerular and tubulointerstitial lesion formation. In addition, up-regulation of MIF expression by parietal epithelial cells was associated with macrophage accumulation within Bowman's space and crescent formation. Combined in situ hybridization and immunostaining also demonstrated MIF expression by macrophages, T cells, and fibroblast-like cells within renal lesions. In conclusion, these data provide the first demonstration that renal epithelial cells are a major source of MIF in both normal and diseased kidney. Furthermore, the up-regulation of MIF expression may play an important role in macrophage accumulation and progressive renal injury in rat crescentic glomerulonephritis.

Granuloma is a specialized form of inflammatory reaction featuring focal macrophage and T-cell accumulation and multinucleated giant cell formation. It is widely held that macrophage accumulation within granulomatous lesions results from recruitment of blood monocytes, whereas proliferation of monocyte/macrophages makes little contribution to this process. The present study of macrophage proliferation within immunologically induced granulomas in rat experimental Goodpasture's syndrome challenges the conventional view. In this disease, granulomatous lesions in the kidney and lung contained 60 to 70% macrophages of an ED1+ED2-ED3-blood monocyte phenotype. However, double immunohistochemistry showed that up to 75% of ED1+ macrophages within granulomatous lesions were proliferating on the basis of proliferating cell nuclear antigen expression and bromodeoxyuridine incorporation. In contrast, no proliferating cell nuclear antigen expression or bromodeoxyuridine incorporations was detected in blood monocytes, indicating that proliferation of ED1+ED2-ED3- cells was a localized event within granulomatous lesions. A second finding of note was that almost all ( > 95%) nuclei within multinucleated giant cells were positive for proliferating cell nuclear antigen, but these nuclei lacked bromodeoxyuridine incorporation. This suggests a novel mechanism of multinucleated giant cell formation involving fusion of macrophages in G1 phase, which then halts progression into S phase of the cell cycle. In conclusion, this study has found that local macrophage proliferation plays an important role in the pathogenesis of granuloma formation.

The functional importance of ICAM-1 and its ligands, the beta 2-integrins, in leukocytic accumulation in pulmonary injury has been recently demonstrated in experimental models of lung disease. However, the exact location of these adhesion molecules remains unknown. In the current study we have used immunogold ultrastructural techniques to define the precise location of ICAM-1 in the lung and its interaction with beta 2-integrin expressing leukocytes in the early stages of experimental Goodpasture's (GP) syndrome in the rat. In normal animals there is strong constitutive ICAM-1 expression on the luminal surface of the alveolar epithelium that is confined to type I cells and completely absent from type II cells. Constitutive expression of ICAM-1 on the pulmonary capillary endothelium is comparatively weak. In GP syndrome there is an increase in ICAM-1 expression, which is still confined to the alveolar type I epithelial cells and capillary endothelium. This is associated with an early (1.5 hours) influx of CD18 expressing polymorphonuclear leukocytes, which are seen migrating into alveoli and the pulmonary interstitium. There is a later (6-12 hours) influx of CD11a/CD18 expressing macrophages which are present in the interstitium and in large numbers in the alveolar spaces, where they are very closely apposed to and adherent to the alveolar epithelium. This is the first study to demonstrate the precise ultrastructural location of ICAM-1 in the normal rat lung and in disease. In vivo administered antibody to ICAM-1 gains access to the extravascular sites within the lung, in particular the surface of alveolar type I epithelial cells, and this raises the possibility that beneficial effects of such antibodies may extend beyond their ability to inhibit interactions between leukocytes and endothelial cells.