Sethu M. Madhavan

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.

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.

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.

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.

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 APOL1s 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.

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.