Effects of Proximal Tubule Shortening on Protein Excretion in a Lowe Syndrome Model

Abstract

BackgroundLowe syndrome (LS) is an X-linked recessive disorder caused by mutations in OCRL, which encodes the enzyme OCRL. Symptoms of LS include proximal tubule (PT) dysfunction typically characterized by low molecular weight proteinuria, renal tubular acidosis (RTA), aminoaciduria, and hypercalciuria. How mutant OCRL causes these symptoms isn’t clear.MethodsWe examined the effect of deleting OCRL on endocytic traffic and cell division in newly created human PT CRISPR/Cas9 OCRL knockout cells, multiple PT cell lines treated with OCRL-targeting siRNA, and in orcl-mutant zebrafish.ResultsOCRL-depleted human cells proliferated more slowly and about 10% of them were multinucleated compared with fewer than 2% of matched control cells. Heterologous expression of wild-type, but not phosphatase-deficient, OCRL prevented the accumulation of multinucleated cells after acute knockdown of OCRL but could not rescue the phenotype in stably edited knockout cell lines. Mathematic modeling confirmed that reduced PT length can account for the urinary excretion profile in LS. Both ocrl mutant zebrafish and zebrafish injected with ocrl morpholino showed truncated expression of megalin along the pronephric kidney, consistent with a shortened S1 segment.ConclusionsOur data suggest a unifying model to explain how loss of OCRL results in tubular proteinuria as well as the other commonly observed renal manifestations of LS. We hypothesize that defective cell division during kidney development and/or repair compromises PT length and impairs kidney function in LS patients.

Predicting the protein composition of human urine in normal and pathological states: Quantitative description based on Dent1 disease (CLCN5 mutation)

“…These values are similar to previous estimates. Lazzara & Deen (2007) estimated the V max of albumin as 2.75 fmol s -1 mm -2 (0.18 ng s -1 mm -2 ) in humans and Gliozzi et al (2020) estimated the value as 0.60 fmol s -1 mm -2 (0.039 ng s -1 mm -2 ); they assumed the same value on a mass basis for low molecular weight proteins (LMWP) and albumin. Figure 2 shows the predicted molar flow profiles of albumin plus α 1 -m, and RBP4 plus β 2 -m along the PT.…”

“…If Dent1 disease almost completely abolishes endocytosis by the PT, and assuming a GFR of 166 L day -1 (Denic et al 2019), the glomerular filtrate concentrations of Gliozzi et al yield a 24 h excretion of ∼3.2 g of albumin and 10.0 g of LMWP, which are much higher values than those associated with FS (Norden et al 2001;Bockenhauer et al 2008). There is evidence suggesting that the rat concentration values used by Gliozzi et al (2020) may be species-specific (Bolton et al 1976;Gomez-Baena et al 2014).…”

“…However, their assumptions about protein concentrations in the glomerular filtrate differ from ours. For their human model, Gliozzi et al (2020) used values derived from micropuncture determinations in the rat: 21.7 μg mL −1 for albumin and 67.3 μg mL −1 for LMWP. Our own values from human Dent1 disease are 3.7 μg mL −1 (albumin) and 5.1 μg mL −1 (sum of β 2 -m, RBP4, and α 1 -m), respectively (Table 1).…”

“…These two pathways mediate protein endocytosis so that only some 0.025% and 1.15% of the amount of filtered proteins, respectively, appear in urine. A similar distinction was made recently by Gliozzi et al (2020). Our description incorporates aspects of both the Lazzara & Deen (2007) and the Gliozzi et al (2020) models; we consider that it is more comprehensive, gives different predictions in humans and, critically, is validated in two non-genetic clinical scenarios.…”

Targeting PIs to improve PTs:a new approach to treating genetic diseases of the proximal tubule

“…4,5 Other factors, including a shortened PT resulting from impaired cell division, have also been suggested to contribute to the proteinuria and ion homeostasis defects characteristic of patients with LS. 6 Complexities in generating a mouse model for LS that accurately recapitulates the human disease likely contribute to this uncertainty, as knockout of Ocrl alone does not result in low molecular weight proteinuria. This is likely due to the abundant expression in mice of Inpp5b, another inositol polyphosphatase that contributes to PI(4,5)P2 levels in endocytic compartments.…”