Development of higher rates of nondiabetic glomerulosclerosis (GS) in African Americans has been attributed to two coding sequence variants (G1 and G2) in the APOL1 gene. To date, the cellular function and the role of APOL1 variants (Vs) in GS are still unknown. In this study, we examined the effects of overexpressing wild-type (G0) and kidney disease risk variants (G1 and G2) of APOL1 in human podocytes using a lentivirus expression system. Interestingly, G0 inflicted podocyte injury only at a higher concentration; however, G1 and G2 promoted moderate podocyte injury at lower and higher concentrations. APOL1Vs expressing podocytes displayed diffuse distribution of both Lucifer yellow dye and cathepsin L as manifestations of enhanced lysosomal membrane permeability (LMP). Chloroquine attenuated the APOL1Vs-induced increase in podocyte injury, consistent with targeting lysosomes. The chloride channel blocker DIDS prevented APOL1Vs- induced injury, indicating a role for chloride influx in osmotic swelling of lysosomes. Direct exposure of noninfected podocytes with conditioned media from G1- and G2-expressing podocytes also induced injury, suggesting a contributory role of the secreted component of G1 and G2 as well. Adverse host factors (AHFs) such as hydrogen peroxide, hypoxia, TNF-α, and puromycin aminonucleoside augmented APOL1- and APOL1Vs-induced podocyte injury, while the effect of human immunodeficiency virus (HIV) on podocyte injury was overwhelming under conditions of APOLVs expression. We conclude that G0 and G1 and G2 APOL1 variants have the potential to induce podocyte injury in a manner which is further augmented by AHFs, with HIV infection being especially prominent.

FSGS is characterized by segmental scarring of the glomerulus and is a leading cause of kidney failure. Identification of genes causing FSGS has improved our understanding of disease mechanisms and points to defects in the glomerular epithelial cell, the podocyte, as a major factor in disease pathogenesis. Using a combination of genome-wide linkage studies and whole-exome sequencing in a kindred with familial FSGS, we identified a missense mutation R431C in anillin (ANLN), an F-actin binding cell cycle gene, as a cause of FSGS. We screened 250 additional families with FSGS and found another variant, G618C, that segregates with disease in a second family with FSGS. We demonstrate upregulation of anillin in podocytes in kidney biopsy specimens from individuals with FSGS and kidney samples from a murine model of HIV-1–associated nephropathy. Overexpression of R431C mutant ANLN in immortalized human podocytes results in enhanced podocyte motility. The mutant anillin displays reduced binding to the slit diaphragm–associated scaffold protein CD2AP. Knockdown of the ANLN gene in zebrafish morphants caused a loss of glomerular filtration barrier integrity, podocyte foot process effacement, and an edematous phenotype. Collectively, these findings suggest that anillin is important in maintaining the integrity of the podocyte actin cytoskeleton.

ANG II has been shown to modulate kidney cell growth and contribute to the pathobiology of glomerulosclerosis. Glomerular visceral epithelial cell (GEC) injury or loss is considered to play a pivotal role in the initiation and progression of glomerulosclerosis. In the present study, we investigated the effect of ANG II on GEC apoptosis. Rat GECs were incubated with increasing doses of ANG II for variable time periods. Apoptosis was evaluated by cell nucleus staining and DNA fragmentation assay. ANG II induced GEC apoptosis in a dose- and time-dependent manner. The proapoptotic effect was attenuated by the ANG II receptor type 1 antagonist losartan or the ANG II receptor type 2 antagonist PD-123319 and was completely blocked by incubation with the combined antagonists. Moreover, ANG II stimulated transforming growth factor (TGF)-β1 production as measured by ELISA. GECs exposed to TGF-β1 demonstrated a dose- and time-dependent increase in apoptosis. ANG II-induced apoptosis was significantly inhibited by addition of anti-TGF-β1 antibody. ANG II also upregulated the expression of Fas, FasL, and Bax and downregulated the expression of Bcl-2 in GECs. These studies suggest that ANG II induces GEC apoptosis by a mechanism involving TGF-β1 expression that may, importantly, contribute to the pathogenesis of glomerulosclerosis.

Laboratory data indicate that morphine decreases the number of peritoneal and alveolar macrophages (Mφ) and compromises their phagocytic capability for immune complexes and bacteria. We hypothesize that morphine decreases the number of, as well as compromises the phagocytic capability of, Mφ by programming their death. We studied the effect of morphine on Mφ apoptosis in vivo as well as in vitro. Peritoneal Mφ harvested from morphine-treated rats showed DNA fragmentation. Morphine enhanced murine Mφ (J 774.16) apoptosis in a dose-dependent manner. Human monocytes treated with morphine showed a classic ladder pattern in gel electrophoretic and end-labeling studies. Morphine promoted nitric oxide (NO) production both under basal and LPS-activated states. NG-nitro-l-arginine methyl ester (l-NAME) and NG-monomethyl-l-arginine monoacetate (l-NMMA), inhibitors of NO synthase, attenuated the morphine-induced generation of NO by Mφ. Morphine also enhanced Mφ mRNA expression of inducible NO synthase (iNOS). Since morphine-induced Mφ apoptosis was inhibited by L-NAME and L-NMMA, it appears that morphine-induced Mφ apoptosis may be mediated through the generation of NO. Morphine promoted the synthesis of Bax and p53 proteins by Mφ. Moreover, IL-converting enzyme (ICE)-1 inhibitor attenuated morphine-induced Mφ apoptosis. These studies suggest that morphine activates the induction phase of the apoptotic pathway through accumulation of p53. The effector phase of morphine-induced apoptosis appears to proceed through the accumulation of Bax and activation of ICE-1. The present study provides a basis for a hypothesis that morphine may be directly compromising immune function by promoting Mφ apoptosis in patients with opiate addiction.

ANG II has been demonstrated to play a role in the progression of tubulointerstial injury. We studied the direct effect of ANG II on apoptosis of cultured rat renal proximal tubular epithelial cells (RPTECs). ANG II promoted RPTEC apoptosis in a dose- and time-dependent manner. This effect of ANG II was attenuated by anti-transforming growth factor (TGF)-β antibody. Moreover, TGF-β triggered RPTEC apoptosis in a dose-dependent manner. ANG II also enhanced RPTEC expression of Fas and Fas ligand (FasL); furthermore, anti-FasL antibody attenuated ANG II-induced RPTEC apoptosis. In addition, ANG II increased RPTEC expression of Bax, a cell death protein. Both ANG II type 1 (AT₁) and type 2 (AT₂) receptor blockers inhibited ANG II-induced RPTEC apoptosis. SB-202190, an inhibitor of p38 MAPK phosphorylation, and caspase-3 inhibitor also attenuated ANG II-induced RPTEC apoptosis. ANG II enhanced RPTEC heme oxygenase (HO)-1 expression. Interestingly, pretreatment with hemin as well as curcumin (inducers of HO-1) inhibited the ANG II-induced tubular cell apoptosis; conversely, pretreatment with zinc protoporphyrin, an inhibitor of HO-1 expression, promoted the effect of ANG II. These results suggest that ANG II-induced apoptosis is mediated via both AT₁and AT₂receptors through the generation of TGF-β, followed by the transcription of cell death genes such as Fas, FasL, and Bax. Modulation of tubular cell expression of HO-1 has an inverse relationship with the ANG II-induced tubular cell apoptosis.