Zinc finger protein 24-dependent transcription factor SOX9 up-regulation protects tubular epithelial cells during acute kidney injury.

Transcriptional profiling studies have identified several protective genes upregulated in tubular epithelial cells during acute kidney injury (AKI). Identifying upstream transcriptional regulators could lead to the development of therapeutic strategies augmenting the repair processes. SOX9 is a transcription factor controlling cell-fate during embryonic development and adult tissue homeostasis in multiple organs including the kidneys. SOX9 expression is low in adult kidneys; however, stress conditions can trigger its transcriptional upregulation in tubular epithelial cells. SOX9 plays a protective role during the early phase of AKI and facilitates repair during the recovery phase. To identify the upstream transcriptional regulators that drive SOX9 upregulation in tubular epithelial cells, we used an unbiased transcription factor screening approach. Preliminary screening and validation studies show that zinc finger protein 24 (ZFP24) governs SOX9 upregulation in tubular epithelial cells. ZFP24, a Cys2-His2 (C2H2) zinc finger protein, is essential for oligodendrocyte maturation and myelination; however, its role in the kidneys or in SOX9 regulation remains unknown. Here, we found that tubular epithelial ZFP24 gene ablation exacerbated ischemia, rhabdomyolysis, and cisplatin-associated AKI. Importantly, ZFP24 gene deletion resulted in suppression of SOX9 upregulation in injured tubular epithelial cells. Chromatin immunoprecipitation and promoter luciferase assays confirmed that ZFP24 bound to a specific site in both murine and human SOX9 promoters. Importantly, CRISPR/Cas9-mediated mutation in the ZFP24 binding site in the SOX9 promoter in vivo led to suppression of SOX9 upregulation during AKI. Thus, our findings identify ZFP24 as a critical stress-responsive transcription factor protecting tubular epithelial cells through SOX9 upregulation.

Essential role of Wtip in mouse development and maintenance of the glomerular filtration barrier.

Wilms' tumor interacting protein (Wtip) has been implicated in cell junction assembly and cell differentiation and interacts with proteins in the podocyte slit diaphragm, where it regulates podocyte phenotype. To define Wtip expression and function in the kidney, we created a Wtip-deleted mouse model using ß-galactosidase-neomycin (ß-geo) gene trap technology. Wtip gene trap mice were embryonic lethal, suggesting additional developmental roles outside kidney function. Using ß-geo heterozygous and normal mice, Wtip expression was identified in the developing kidneys, heart, and eyes. In the kidney, expression was restricted to podocytes, which appeared initially at the capillary loop stage coinciding with terminal podocyte differentiation. Heterozygous mice had an expected lifespan and showed no evidence of proteinuria or glomerular pathology. However, heterozygous mice were more susceptible to glomerular injury than wild-type littermates and developed more significant and prolonged proteinuria in response to lipopolysaccharide or adriamycin. In normal human kidneys, WTIP expression patterns were consistent with observations in mice and were lost in glomeruli concurrent with loss of synaptopodin expression in disease. Mechanistically, we identified the Rho guanine nucleotide exchange factor 12 (ARHGEF12) as a binding partner for WTIP. ARHGEF12 was expressed in human podocytes and formed high-affinity interactions through their LIM- and PDZ-binding domains. Our findings suggest that Wtip is essential for early murine embryonic development and maintaining normal glomerular filtration barrier function, potentially regulating slit diaphragm and foot process function through Rho effector proteins.NEW & NOTEWORTHY This study characterized dynamic expression patterns of Wilms' tumor interacting protein (Wtip) and demonstrates the novel role of Wtip in murine development and maintenance of the glomerular filtration barrier.

Acute kidney injury in patients treated with immune checkpoint inhibitors.

BACKGROUND: Immune checkpoint inhibitor-associated acute kidney injury (ICPi-AKI) has emerged as an important toxicity among patients with cancer. METHODS: We collected data on 429 patients with ICPi-AKI and 429 control patients who received ICPis contemporaneously but who did not develop ICPi-AKI from 30 sites in 10 countries. Multivariable logistic regression was used to identify predictors of ICPi-AKI and its recovery. A multivariable Cox model was used to estimate the effect of ICPi rechallenge versus no rechallenge on survival following ICPi-AKI. RESULTS: ICPi-AKI occurred at a median of 16 weeks (IQR 8-32) following ICPi initiation. Lower baseline estimated glomerular filtration rate, proton pump inhibitor (PPI) use, and extrarenal immune-related adverse events (irAEs) were each associated with a higher risk of ICPi-AKI. Acute tubulointerstitial nephritis was the most common lesion on kidney biopsy (125/151 biopsied patients [82.7%]). Renal recovery occurred in 276 patients (64.3%) at a median of 7 weeks (IQR 3-10) following ICPi-AKI. Treatment with corticosteroids within 14 days following ICPi-AKI diagnosis was associated with higher odds of renal recovery (adjusted OR 2.64; 95% CI 1.58 to 4.41). Among patients treated with corticosteroids, early initiation of corticosteroids (within 3 days of ICPi-AKI) was associated with a higher odds of renal recovery compared with later initiation (more than 3 days following ICPi-AKI) (adjusted OR 2.09; 95% CI 1.16 to 3.79). Of 121 patients rechallenged, 20 (16.5%) developed recurrent ICPi-AKI. There was no difference in survival among patients rechallenged versus those not rechallenged following ICPi-AKI. CONCLUSIONS: Patients who developed ICPi-AKI were more likely to have impaired renal function at baseline, use a PPI, and have extrarenal irAEs. Two-thirds of patients had renal recovery following ICPi-AKI. Treatment with corticosteroids was associated with improved renal recovery.

Lack of APOL1 in proximal tubules of normal human kidneys and proteinuric APOL1 transgenic mouse kidneys.

The mechanism of pathogenesis associated with APOL1 polymorphisms and risk for non-diabetic chronic kidney disease (CKD) is not fully understood. Prior studies have minimized a causal role for the circulating APOL1 protein, thus efforts to understand kidney pathogenesis have focused on APOL1 expressed in renal cells. Of the kidney cells reported to express APOL1, the proximal tubule expression patterns are inconsistent in published reports, and whether APOL1 is synthesized by the proximal tubule or possibly APOL1 protein in the blood is filtered and reabsorbed by the proximal tubule remains unclear. Using both protein and mRNA in situ methods, the kidney expression pattern of APOL1 was examined in normal human and APOL1 bacterial artificial chromosome transgenic mice with and without proteinuria. APOL1 protein and mRNA was detected in podocytes and endothelial cells, but not in tubular epithelia. In the setting of proteinuria, plasma APOL1 protein did not appear to be filtered or reabsorbed by the proximal tubule. A side-by-side examination of commercial antibodies used in prior studies suggest the original reports of APOL1 in proximal tubules likely reflects antibody non-specificity. As such, APOL1 expression in podocytes and endothelia should remain the focus for mechanistic studies in the APOL1-mediated kidney diseases.

The Relationship between APOL1 Structure and Function: Clinical Implications.

Common variants in the APOL1 gene are associated with an increased risk of nondiabetic kidney disease in individuals of African ancestry. Mechanisms by which APOL1 variants mediate kidney disease pathogenesis are not well understood. Amino acid changes resulting from the kidney disease-associated APOL1 variants alter the three-dimensional structure and conformational dynamics of the C-terminal α-helical domain of the protein, which can rationalize the functional consequences. Understanding the three-dimensional structure of the protein, with and without the risk variants, can provide insights into the pathogenesis of kidney diseases mediated by APOL1 variants.

ApoL1 Overexpression Drives Variant-Independent Cytotoxicity.

Coding variants in the APOL1 gene are associated with kidney diseases in African ancestral populations; yet, the underlying biologic mechanisms remain uncertain. Variant-dependent autophagic and cytotoxic cell death have been proposed as pathogenic pathways mediating kidney injury. To examine this possibility, we conditionally expressed APOL1-G0 (reference), -G1, and -G2 (variants) using a tetracycline-regulated system in HEK293 cells. Autophagy was monitored biochemically and cell death was measured using multiple assays. We measured intracellular Na+ and K+ content with atomic absorption spectroscopy and APOL1-dependent currents with whole-cell patch clamping. Neither reference nor variant APOL1s induced autophagy. At high expression levels, APOL1-G0, -G1, and -G2 inserted into the plasma membrane and formed pH-sensitive cation channels, causing collapse of cellular Na+ and K+ gradients, phosphorylation of p38 mitogen-activated protein kinase, and cell death, without variant-dependent differences. APOL1-G0 and -G2 exhibited similar channel properties in whole-cell patch clamp experiments. At low expression levels, neither reference nor variant APOL1s localized on the plasma membrane, Na+ and K+ gradients were maintained, and cells remained viable. Our results indicate that APOL1-mediated pore formation is critical for the trypanolytic activity of APOL1 and drives APOL1-mediated cytotoxicity in overexpression systems. The absence of cytotoxicity at physiologic expression levels suggests variant-dependent intracellular K+ loss and cytotoxicity does not drive kidney disease progression.

Kidney Proximal Tubule Lipoapoptosis Is Regulated by Fatty Acid Transporter-2 (FATP2).

Albuminuria and tubular atrophy are among the highest risks for CKD progression to ESRD. A parsimonious mechanism involves leakage of albumin-bound nonesterified fatty acids (NEFAs) across the damaged glomerular filtration barrier and subsequent reabsorption by the downstream proximal tubule, causing lipoapoptosis. We sought to identify the apical proximal tubule transporter that mediates NEFA uptake and cytotoxicity. We observed transporter-mediated uptake of fluorescently labeled NEFA in cultured proximal tubule cells and microperfused rat proximal tubules, with greater uptake from the apical surface than from the basolateral surface. Protein and mRNA expression analyses revealed that kidney proximal tubules express transmembrane fatty acid transporter-2 (FATP2), encoded by Slc27a2, but not the other candidate transporters CD36 and free fatty acid receptor 1. Kidney FATP2 localized exclusively to proximal tubule epithelial cells along the apical but not the basolateral membrane. Treatment of mice with lipidated albumin to induce proteinuria caused a decrease in the proportion of tubular epithelial cells and an increase in the proportion of interstitial space in kidneys from wild-type but not Slc27a2-/- mice. Ex vivo microperfusion and in vitro experiments with NEFA-bound albumin at concentrations that mimic apical proximal tubule exposure during glomerular injury revealed significantly reduced NEFA uptake and palmitate-induced apoptosis in microperfused Slc27a2-/- proximal tubules and Slc27a2-/- or FATP2 shRNA-treated proximal tubule cell lines compared with wild-type or scrambled oligonucleotide-treated cells, respectively. We conclude that FATP2 is a major apical proximal tubule NEFA transporter that regulates lipoapoptosis and may be an amenable target for the prevention of CKD progression.

APOL1 variants change C-terminal conformational dynamics and binding to SNARE protein VAMP8.

APOL1 variants in African populations mediate resistance to trypanosomal infection but increase risk for kidney diseases through unknown mechanisms. APOL1 is expressed in glomerular podocytes and does not vary with underlying kidney disease diagnoses or APOL1 genotypes, suggesting that the kidney disease-associated variants dysregulate its function rather than its localization or abundance. Structural homology searches identified vesicle-associated membrane protein 8 (VAMP8) as an APOL1 protein interactor. VAMP8 colocalizes with APOL1 in the podocyte, and the APOL1:VAMP8 interaction was confirmed biochemically and with surface plasmon resonance. APOL1 variants attenuate this interaction. Computational modeling of APOL1's 3-dimensional structure, followed by molecular dynamics simulations, revealed increased motion of the C-terminal domain of reference APOL1 compared with either variant, suggesting that the variants stabilize a closed or autoinhibited state that diminishes protein interactions with VAMP8. Changes in ellipticity with increasing urea concentrations, as assessed by circular dichroism spectroscopy, showed higher conformational stability of the C-terminal helix of the variants compared with the reference protein. These results suggest that reference APOL1 interacts with VAMP8-coated vesicles, a process attenuated by variant-induced reduction in local dynamics of the C-terminal. Disordered vesicular trafficking in the podocyte may cause injury and progressive chronic kidney diseases in susceptible African Americans subjects.

APOL1-G0 or APOL1-G2 Transgenic Models Develop Preeclampsia but Not Kidney Disease.

APOL1 risk variants are associated with kidney disease in blacks, but the mechanisms of renal injury associated with APOL1 risk variants are unknown. Because APOL1 is unique to humans and some primates, we created transgenic (Tg) mice using the promoter of nephrin-encoding Nphs1 to express the APOL1 reference sequence (G0) or the G2 risk variant in podocytes, establishing Tg lines with a spectrum of APOL1 expression levels. Podocytes from Tg-G0 and Tg-G2 mice did not undergo necrosis, apoptosis, or autophagic cell death in vivo, even in lines with highly expressed transgenes. Further, Tg-G0 and Tg-G2 mice did not develop kidney pathology, proteinuria, or azotemia as of 300 days of age. However, by 200 days of age, Tg-G2 mice had significantly lower podocyte density than age-matched WT and Tg-G0 mice had, a difference that was not evident at weaning. Notably, a pregnancy-associated phenotype that encompassed eclampsia, preeclampsia, fetal/neonatal deaths, and small litter sizes occurred in some Tg-G0 mice and more severely in Tg-G2 mice. Similar to human placenta, placentas of Tg mice expressed APOL1. Overall, these results suggest podocyte depletion could predispose individuals with APOL1 risk genotypes to kidney disease in response to a second stressor, and add to other published evidence associating APOL1 expression with preeclampsia.

The biology of APOL1 with insights into the association between APOL1 variants and chronic kidney disease.

Recent studies have identified genetic variants in APOL1 that may contribute to the increased incidence of kidney disease in populations with African ancestry. Here, we review the biology of APOL1 present in the circulation and localized to the kidney as it may contribute to the pathogenesis of APOL1-associated kidney disease.

Plasma apolipoprotein L1 levels do not correlate with CKD.

Polymorphisms in APOL1 are associated with CKD, including HIV-related CKD, in individuals of African ancestry. The apolipoprotein L1 (APOL1) protein circulates and is localized in kidney cells, but the contribution of APOL1 location to CKD pathogenesis is unclear. We examined associations of plasma APOL1 levels with plasma cytokine levels, dyslipidemia, and APOL1 genotype in a nested case-control study (n=270) of HIV-infected African Americans enrolled in a multicenter prospective observational study. Patients were designated as having CKD when estimated GFR (eGFR) decreased to <60 ml/min per 1.73 m(2) (eGFR<60 cohort) or protein-to-creatinine ratios became >3.5 g/g (nephrotic proteinuria cohort). Circulating APOL1 levels did not associate with APOL1 genotype, CKD status, or levels of proinflammatory cytokines, but did correlate with fasting cholesterol, LDL cholesterol, and triglyceride levels. At ascertainment, CKD-associated polymorphisms (risk variants) in APOL1 associated with the eGFR<60 cohort, but not the nephrotic-range proteinuria cohort. Of note, in both the eGFR<60 and nephrotic proteinuria cohorts, CKD cases with two APOL1 risk variants had significant declines in eGFR over a median of 4 years compared with individuals with one or no risk variants. APOL1 risk genotype was not associated with changes in proteinuria. Higher circulating proinflammatory cytokine levels were independently associated with CKD but not APOL1 genotype. In conclusion, the function of variant APOL1 proteins derived from circulation or synthesized in the kidney, but not the level of circulating APOL1, probably mediates APOL1-associated kidney disease in HIV-infected African Americans.

WT1-interacting protein (Wtip) regulates podocyte phenotype by cell-cell and cell-matrix contact reorganization.

Podocytes respond to environmental cues by remodeling their slit diaphragms and cell-matrix adhesive junctions. Wt1-interacting protein (Wtip), an Ajuba family LIM domain scaffold protein expressed in the podocyte, coordinates cell adhesion changes and transcriptional responses to regulate podocyte phenotypic plasticity. We evaluated effects of Wtip on podocyte cell-cell and cell-matrix contact organization using gain-of- and loss-of-function methods. Endogenous Wtip targeted to focal adhesions in adherent but isolated podocytes and then shifted to adherens junctions after cells made stable, homotypic contacts. Podocytes with Wtip knockdown (shWtip) adhered but failed to spread normally. Noncontacted shWtip podocytes did not assemble actin stress fibers, and their focal adhesions failed to mature. As shWtip podocytes established cell-cell contacts, stable adherens junctions failed to form and F-actin structures were disordered. In shWtip cells, cadherin and ß-catenin clustered in irregularly distributed spots that failed to laterally expand. Cell surface biotinylation showed diminished plasma membrane cadherin, ß-catenin, and α-catenin in shWtip podocytes, although protein expression was similar in shWtip and control cells. Since normal actin dynamics are required for organization of adherens junctions and focal adhesions, we determined whether Wtip regulates F-actin assembly. Undifferentiated podocytes did not elaborate F-actin stress fibers, but when induced to overexpress WTIP, formed abundant stress fibers, a process blocked by the RhoA inhibitor C3 toxin and a RhoA kinase inhibitor. WTIP directly interacted with Rho guanine nucleotide exchange factor (GEF) 12 (Arhgef12), a RhoA-specific GEF enriched in the glomerulus. In conclusion, stable assembly of podocyte adherens junctions and cell-matrix contacts requires Wtip, a process that may be mediated by spatiotemporal regulation of RhoA activity through appropriate targeting of Arhgef12.

APOL1 localization in normal kidney and nondiabetic kidney disease.

In patients of African ancestry, genetic variants in APOL1, which encodes apolipoprotein L1, associate with the nondiabetic kidney diseases, focal segmental glomerulosclerosis (FSGS), HIV-associated nephropathy (HIVAN), and hypertensive nephropathy. Understanding the renal localization of APOL1 may provide clues that will ultimately help elucidate the mechanisms by which APOL1 variants promote nephropathy. Here, we used immunohistology to examine APOL1 localization in normal human kidney sections and in biopsies demonstrating either FSGS (n = 8) or HIVAN (n = 2). Within normal glomeruli, APOL1 only localized to podocytes. Compared with normal glomeruli, fewer cells stained for APOL1 in FSGS and HIVAN glomeruli, even when expression of the podocyte markers GLEPP1 and synaptopodin appeared normal. APOL1 localized to proximal tubular epithelia in normal kidneys, FSGS, and HIVAN. We detected APOL1 in the arteriolar endothelium of normal and diseased kidney sections. Unexpectedly, in both FSGS and HIVAN but not normal kidneys, the media of medium artery and arterioles contained a subset of α-smooth muscle actin-positive cells that stained for APOL1. Comparing the renal distribution of APOL1 in nondiabetic kidney disease to normal kidney suggests that a previously unrecognized arteriopathy may contribute to disease pathogenesis in patients of African ancestry.

Out on a LIM: chronic kidney disease, podocyte phenotype and the Wilm's tumor interacting protein (WTIP).

Normal function of the glomerular filtration barrier requires wild-type differentiation of the highly specialized glomerular epithelial cell, the podocyte. Podocytes express three distinct domains, consisting of a cell body, primary processes, and secondary foot processes (FP). These FP express slit diaphragms, which are highly specialized cell-cell contacts critical for filtration-barrier function. Foot processes are dynamic structures that reorganize within minutes through actin cytoskeletal rearrangement. Glomerular diseases are characterized by a persistent simplification in podocyte domain structure with loss of FP, a phenotype described as FP effacement. The generation of such phenotypic plasticity requires that signaling pathways in subcellular compartments be integrated dynamically for a cell to respond appropriately to information flow from its microenvironment. We have identified a LIM-domain-containing protein, Wilm's tumor interacting protein (WTIP), that regulates podocyte actin dynamics to maintain stable cell contacts. After glomerular injury, the WTIP molecule shuttles to the podocyte nucleus in response to changes in slit-diaphragm assembly, and changes gene transcription to permit podocyte remodeling. Defining regulatory pathways of podocyte differentiation identifies novel, druggable targets for chronic kidney diseases characterized by glomerular scarring.

Individuals with mutations in XPNPEP3, which encodes a mitochondrial protein, develop a nephronophthisis-like nephropathy.

The autosomal recessive kidney disease nephronophthisis (NPHP) constitutes the most frequent genetic cause of terminal renal failure in the first 3 decades of life. Ten causative genes (NPHP1-NPHP9 and NPHP11), whose products localize to the primary cilia-centrosome complex, support the unifying concept that cystic kidney diseases are "ciliopathies". Using genome-wide homozygosity mapping, we report here what we believe to be a new locus (NPHP-like 1 [NPHPL1]) for an NPHP-like nephropathy. In 2 families with an NPHP-like phenotype, we detected homozygous frameshift and splice-site mutations, respectively, in the X-prolyl aminopeptidase 3 (XPNPEP3) gene. In contrast to all known NPHP proteins, XPNPEP3 localizes to mitochondria of renal cells. However, in vivo analyses also revealed a likely cilia-related function; suppression of zebrafish xpnpep3 phenocopied the developmental phenotypes of ciliopathy morphants, and this effect was rescued by human XPNPEP3 that was devoid of a mitochondrial localization signal. Consistent with a role for XPNPEP3 in ciliary function, several ciliary cystogenic proteins were found to be XPNPEP3 substrates, for which resistance to N-terminal proline cleavage resulted in attenuated protein function in vivo in zebrafish. Our data highlight an emerging link between mitochondria and ciliary dysfunction, and suggest that further understanding the enzymatic activity and substrates of XPNPEP3 will illuminate novel cystogenic pathways.