Tissue Culture Models of Acute Kidney Injury: From Tubule Cells to Human Kidney Organoids

Acute kidney injury (AKI) affects approximately 13.3 million people around the world each year, causing chronic kidney disease and/or mortality. The mammalian kidney cannot generate new nephrons after postnatal renal damage and regenerative therapies for AKI are not available. Human kidney tissue culture systems can complement animal models of AKI and/or address some of their limitations. Donor-derived somatic cells, such as renal tubule epithelial cells or cell lines (RPTEC/hTERT, ciPTEC, HK-2, Nki-2, and CIHP-1), have been used for decades to permit drug toxicity screening and studies into potential AKI mechanisms. However, tubule cell lines do not fully recapitulate tubular epithelial cell properties in situ when grown under classic tissue culture conditions. Improving tissue culture models of AKI would increase our understanding of the mechanisms, leading to new therapeutics. Human pluripotent stem cells (hPSCs) can be differentiated into kidney organoids and various renal cell types. Injury to human kidney organoids results in renal cell type crosstalk and upregulation of kidney injury biomarkers that are difficult to induce in primary tubule cell cultures. However, current protocols produce kidney organoids that aren't mature and contain off-target cell types. Promising bioengineering techniques, such as bioprinting and "kidney-on-a17 chip" methods, as applied to kidney nephrotoxicity modeling advantages and limitations are discussed. This review explores the mechanisms and detection of AKI in tissue culture, with an emphasis on bioengineered approaches such as human kidney organoid models.

Claudin-10a Deficiency Shifts Proximal Tubular Cl- Permeability to Cation Selectivity via Claudin-2 Redistribution

Background The tight junction proteins claudin-2 and claudin-10a form paracellular cation and anion channels, respectively, and are expressed in the proximal tubule. However, the physiological role of claudin-10a in the kidney has been unclear. Methods To investigate the physiologic role of claudin-10a, we generated claudin-10a-deficient mice; confirmed successful knockout by Southern blot, Western blot, and immunofluorescence staining; and analyzed urine and serum of knockout and wild-type animals. We also used electrophysiologic studies to investigate the functionality of isolated proximal tubules, and studied compensatory regulation by pharmacologic intervention, RNA sequencing analysis, Western blot, immunofluorescence staining, and respirometry. Results Mice deficient in claudin-10a were fertile and without overt phenotypes. Upon knockout, claudin-10a was replaced by claudin-2 in all proximal tubule segments. Electrophysiology showed conversion from paracellular anion preference to cation preference and a loss of paracellular Cl- over HCO3- preference. As a consequence, there was tubular retention of calcium and magnesium, higher urine pH, and mild hypermagnesemia. A comparison of other urine and serum parameters under control conditions and sequential pharmacologic transport inhibition, as well as unchanged fractional lithium excretion, suggested compensative measures in proximal and distal tubular segments. Changes in proximal tubular oxygen handling and differential expression of genes regulating fatty acid metabolism indicated proximal tubular adaptation. Western blot and immunofluorescence revealed alterations in distal tubular transport. Conclusions Claudin-10a is the major paracellular anion channel in the proximal tubule and its deletion causes calcium and magnesium hyperreabsorption by claudin-2 redistribution. Transcellular transport in proximal and distal segments and proximal tubular metabolic adaptation compensate for loss of paracellular anion permeability.

Gentamicin Inhibits Ca2+ Channel TPRV5 and Induces Calciuresis Independent of the Calcium-Sensing Receptor-Claudin-14 Pathway

Background Treatment with the aminoglycoside antibiotic gentamicin can be associated with severe adverse effects, including renal calcium wasting. The underlying mechanism is unknown but it has been proposed to involve activation of the Ca2+-sensing receptor (CaSR) in the thick ascending limb, which would increase expression of claudin-14 (CLDN14) and limit Ca2+ reabsorption. However, no direct evidence for this hypothesis has been presented. Methods We studied the effect of gentamicin in vivo using mouse models with impaired Ca2+ reabsorption in the proximal tubule and the thick ascending limb. We used a Cldn14 promoter luciferase-reporter assay to study CaSR activation and investigated the effect of gentamicin on activity of the distal nephron Ca2+ channel transient potential receptor vanilloid 5 (TPRV5), as determined by patch-clamp in HEK293 cells. Results Gentamicin increased urinary Ca2+ excretion in wild-type mice following acute and chronic administration. This calciuretic effect was unaltered in mice with genetic CaSR overactivation and was present in furosemide-treated animals, whereas the calciuretic effect in Cldn14-/-mice and mice with impaired proximal tubular Ca2+ reabsorption (claudin-2 [CLDN2]-deficient Cldn2-/- mice) was equivalent to that of wild-type mice. In vitro, gentamicin failed to activate the CaSR. In contrast, patch-clamp analysis revealed that gentamicin strongly inhibited rabbit and human TRPV5 activity and that chronic gentamicin administration downregulated distal nephron Ca2+ transporters. Conclusions Gentamicin does not cause hypercalciuria via activation of the CaSR-CLDN14 pathway or by interfering with proximal tubular CLDN2-dependent Ca2+ reabsorption. Instead, gentamicin blocks distal Ca2+ reabsorption by direct inhibition of the Ca2+ channel TRPV5. These findings offer new insights into calcium wasting in patients treated with gentamicin.

Mechanisms and Models of Kidney Tubular Necrosis and Nephron Loss

Understanding nephron loss is a primary strategy for preventing chronic kidney disease (CKD) progression. Death of renal tubular cells may occur by apoptosis during developmental and regenerative processes. However, during acute kidney injury (AKI), the transition of AKI to CKD, sepsis-associated AKI, and kidney transplantation, ferroptosis and necroptosis, two pathways associated with the loss of plasma membrane integrity, kill renal cells. This necrotic type of cell death is associated with an inflammatory response, which is referred to as necroinflammation. Importantly, the necroinflammatory response to cells that die by necroptosis may be fundamentally different from the tissue response to ferroptosis. While mechanisms of ferroptosis and necroptosis have recently been investigated in detail, the cell death propagation during tubular necrosis, although described morphologically, remains incompletely understood. Here, we argue that a molecular switch downstream of tubular necrosis determines nephron regeneration vs. nephron loss, respectively. Unravelling the details of this "switch" must include the inflammatory response to tubular necrosis and regenerative signals potentially controlled by inflammatory cells, including the stimulation of myofibroblasts as the origin of fibrosis. Understanding in detail the molecular switch and the inflammatory responses to tubular necrosis can inform discussion of therapeutic options.

Angiopoietins as Prognostic Markers for Future Kidney Disease and Heart Failure Events After Acute Kidney Injury

Background The mechanisms underlying long-term sequelae following acute kidney injury (AKI) remain unclear. Vessel instability, an early response to endothelial injury, may reflect a shared mechanism and early trigger for chronic kidney disease (CKD) and heart failure. Methods To investigate whether plasma angiopoietins, markers of vessel homeostasis, are associated with CKD progression and heart failure admissions after hospitalization in patients with and without AKI, we conducted a prospective cohort study to analyze the balance between angiopoietin-1 (Angpt-1), which maintains vessel stability, and angiopoietin-2 (Angpt-2), which increases vessel destabilization. Three months after discharge, we evaluated the associations between angiopoietins and development of the primary outcomes of CKD progression and heart failure, as well as the secondary outcome of all-cause mortality 3 months after discharge or later. Results Median age for the 1503 participants was 65.8 years; 746 (50%) had AKI. Compared with the lowest quartile, the highest quartile of the Angpt-1:Angpt-2 ratio was associated with 72% lower risk of CKD progression (adjusted hazard ratio [aHR], 0.28; 95% confidence interval [95% CI], 0.15 to 0.51), 94% lower risk of heart failure (aHR, 0.06; 95% CI, 0.02 to 0.15), and 82% lower risk of mortality (aHR, 0.18; 95% CI, 0.09 to 0.35) for those with AKI. Among those without AKI, the highest quartile of Angpt-1:Angpt-2 ratio was associated with 71% lower risk of heart failure (aHR, 0.29; 95% CI, 0.12 to 0.69) and 68% less mortality (aHR, 0.32; 95% CI, 0.15 to 0.68). There were no associations with CKD progression. Conclusions A higher Angpt-1:Angpt-2 ratio was strongly associated with less CKD progression, heart failure, and mortality in the setting of AKI.

Using Machine Learning to Identify Metabolomic Signatures of Pediatric Chronic Kidney Disease Etiology

BackgroundUntargeted plasma metabolomic profiling combined with machine learning (ML) may lead to discovery of metabolic profiles that inform our understanding of pediatric CKD causes. We sought to identify metabolomic signatures in pediatric CKD based on diagnosis: FSGS, obstructive uropathy (OU), aplasia/dysplasia/hypoplasia (A/D/H), and reflux nephropathy (RN).MethodsUntargeted metabolomic quantification (GC-MS/LC-MS, Metabolon) was performed on plasma from 702 Chronic Kidney Disease in Children study participants (n: FSGS=63, OU=122, A/D/H=109, and RN=86). Lasso regression was used for feature selection, adjusting for clinical covariates. Four methods were then applied to stratify significance: logistic regression, support vector machine, random forest, and extreme gradient boosting. ML training was performed on 80% total cohort subsets and validated on 20% holdout subsets. Important features were selected based on being significant in at least two of the four modeling approaches. We additionally performed pathway enrichment analysis to identify metabolic subpathways associated with CKD cause.ResultsML models were evaluated on holdout subsets with receiver-operator and precision-recall area-under-the-curve, F1 score, and Matthews correlation coefficient. ML models outperformed no-skill prediction. Metabolomic profiles were identified based on cause. FSGS was associated with the sphingomyelin-ceramide axis. FSGS was also associated with individual plasmalogen metabolites and the subpathway. OU was associated with gut microbiome–derived histidine metabolites.ConclusionML models identified metabolomic signatures based on CKD cause. Using ML techniques in conjunction with traditional biostatistics, we demonstrated that sphingomyelin-ceramide and plasmalogen dysmetabolism are associated with FSGS and that gut microbiome–derived histidine metabolites are associated with OU.

Recurrence of Anti-semaphorin-3B Mediated Membranous Nephropathy after Kidney Transplantation

Background: Membranous nephropathy (MN) is rare in pediatric patients, although its diagnosis may be underestimated in children who are responsive to corticosteroid therapy prescribed for a suspicion of minimal change disease. It is most often associated with an autoimmune disease, predominantly lupus. We previously reported the occurrence of early onset membranous nephropathy associated with Semaphorin 3B in 9 children and 2 adults. Methods: Biopsies were performed on native kidney and at 1 and 5 months after transplantation. Semaphorin 3B antigen was detected in immune deposits by immunohistochemistry and confocal microscopy on paraffin-embedded biopsies. Anti-Semaphorin antibodies were detected by Western blot and analyzed sequentially. Results: We report the first case of early recurrence after transplantation in a 7-year old boy who presented with severe nephrotic syndrome and advanced kidney failure. There was no evidence of hereditary or associated autoimmune disease. Abundant, almost coalescent deposits were seen by electron microscopy and bright granular, subepithelial staining was observed for Semaphorin 3B antigen. Western blot analysis of serum revealed antiSemaphorin 3B antibodies. Recurrence of MN occurred 25 days after transplantation and manifested as nephrotic range proteinuria despite conventional immunosuppressive therapy. Kidney biopsies confirmed histological MN recurrence with colocalization of Semaphorin 3B antigen and IgG (1). The patient was treated with rituximab. Anti-Semaphorin 3B antibodies, which were detected at transplantation, were not detected 40 days after rituximab. Conclusion: This case provides evidence that anti-Semaphorin 3B antibodies are pathogenic and should be monitored in patients with membranous nephropathy.

Vitamin C Deficiency Causes Cell Type-Specific Epigenetic Reprogramming and Acute Tubular Necrosis in a Mouse Model

Background: Vitamin C deficiency is found in patients with variable renal diseases. However, the role of vitamin C as an epigenetic regulator in renal homeostasis and pathogenesis remains largely unknown. Methods: We showed that vitamin C deficiency leads to acute tubular necrosis (ATN) using a vitamin C-deficient mouse model (Gulo knock-out). DNA/RNA epigenetic modifications and injured S3 proximal tubule cells were identified in the vitamin C-deficient kidneys using whole-genome bisulfite sequencing, methylated RNA immunoprecipitation sequencing, and single-cell RNA sequencing. Results: Integrated evidence suggested that epigenetic modifications affected the proximal tubule cells and fenestrated endothelial cells, leading to tubule injury and hypoxia through transcriptional regulation. Strikingly, loss of DNA hydroxymethylation and DNA hypermethylation in vitamin C-deficient kidneys preceded the histological sign of tubule necrosis, indicating the causality of vitamin C-induced epigenetic modification in ATN. Consistently, prophylactic supplementation of an oxidation-resistant vitamin C derivative, ascorbyl phosphate magnesium, promoted DNA demethylation and prevented the progression of cisplatin-induced ATN. Conclusions: Vitamin C played a critical role in renal homeostasis and pathogenesis in a mouse model, suggesting vitamin supplementation may be an approach to lower risk of kidney injury.