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Two heterozygous missense variants (G1 and G2) of Apolipoprotein L1 (APOL1) found in individuals of recent African ancestry can attenuate the severity of infection by some forms of Trypanosoma brucei. However, these two variants within a broader African haplotype also increase the risk of kidney disease in Americans of African descent. Although overexpression of either variant G1 or G2 causes multiple pathogenic changes in cultured cells and transgenic mouse models, the mechanism(s) promoting kidney disease remain unclear. Human serum APOL1 kills trypanosomes through its cation channel activity, and cation channel activity of recombinant APOL1 has been reconstituted in lipid bilayers and proteoliposomes. Although APOL1 overexpression increases whole cell cation currents in HEK-293 cells, the ion channel activity of APOL1 has not been assessed in glomerular podocytes, the major site of APOL1-associated kidney diseases. We characterize APOL1-associated whole cell and on-cell cation currents in HEK-293 T-Rex cells and demonstrate partial inhibition of currents by anti-APOL antibodies. We detect in primary human podocytes a similar cation current inducible by interferon-γ (IFNγ) and sensitive to inhibition by anti-APOL antibody as well as by a fragment of T. brucei Serum Resistance-Associated protein (SRA). CRISPR knockout of APOL1 in human primary podocytes abrogates the IFNγ-induced, antibody-sensitive current. Our novel characterization in HEK-293 cells of heterologous APOL1-associated cation conductance inhibited by anti-APOL antibody and our documentation in primary human glomerular podocytes of endogenous IFNγ-stimulated, APOL1-mediated, SRA and anti-APOL-sensitive ion channel activity together support APOL1-mediated channel activity as a therapeutic target for treatment of APOL1-associated kidney diseases.

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The purpose of this study was to elucidate certain aspects of the control of bicarbonate reabsorption in the surface distal tubule of rats made acidotic by ammonium chloride treatment. During acute metabolic acidosis, the effects of sodium delivery, water reabsorption, inhibition of carbonic anhydrase, and addition of the anion channel blocker SITS were determined. In addition, bicarbonate reabsorption was assessed during recovery from metabolic acidosis. Studies during the acute phase of acidosis: Sprague-Dawley rats were made acidotic by ammonium chloride gavage and then prepared for micropuncture. It was hypothesized that distal tubule bicarbonate transport might have components dependent on direct or indirect Na/H exchange, water reabsorption, carbonic anhydrase activity and chloride conductance. To test these hypotheses surface distal tubules were then microperfused in vivo at 8 nl/min with 4 different isoosmotic, bicarbonate containing solutions. These solutions were designed to (1) provide an index of the increased bicarbonate reabsorption evident in this model and assess the effect of the following on bicarbonate reabsorption: (2) sodium replacement with choline; (3) sodium substitution augmented by pharmacologic means (amiloride); (4) inhibition of carbonic anhydrase by acetazolamide. At 8 nl/min, collection of fluid from tubules perfused with all 4 solutions revealed significant bicarbonate reabsorption. Net distal tubule bicarbonate reabsorption was significantly increased in acidotic animals. Perfusion of tubules with sodium free solutions did not decrease this brisk reabsorptive flux, nor did perfusion of sodium free solutions which also contained amiloride. However, perfusion of tubules with the carbonic anhydrase inhibitor DIAMOX significantly decreased bicarbonate reabsorption. This is the first in vivo demonstration of such an effect in the distal tubule of the acidotic rat when bicarbonate load was held constant. The effect of sodium replacement at high perfusion rate was also studied, but again was not found to significantly alter bicarbonate reabsorption, although more bicarbonate was found to be reabsorbed at 25 nl/min than at 8 nl/min. Since some reports have suggested that a chloride conductance may play a role in acidification by proton pumps, the effects of perfusion with a chloride channel blocker was studied. Paired collections at 25 nl/min, with and without SITS, revealed a significant inhibitory effect of SITS. Studies during recovery from metabolic acidosis: Animals allowed to recover from the acidosis exhibited a rebound metabolic alkalosis. It was hypothesized that distal tubule bicarbonate reabsorption might contribute to this apparent overshoot of blood bicarbonate concentration. Tubules perfused 44 hours post gavage exhibited significant bicarbonate reabsorption at 8, 15, and 25 nl/min, despite the concurrent metabolic alkalosis. These are the first reported in vivo data of distal tubule bicarbonate reabsorption during rebound metabolic alkalosis. It is concluded that bicarbonate reabsorption in distal tubules of acidotic rats perfused in vivo at 8 nl/min was not affected by reduced sodium delivery, or water movement, but is partly dependent on carbonic anhydrase activity and may also be partly dependent on chloride permeability. In addition, bicarbonate reabsorption continues during the recovery phase, despite the existence of systemic metabolic alkalosis.