Podocyte ZHX2 expression is important in the development of Focal Segmental Glomerulosclerosis and Minimal Change disease
Authors: Del Nogal Avila Maria Nogal , Blazquez Hector Donoro , Marshall Caroline B , Casado Carmen Avila , Mace Camille E , Clement Lionel C , Chugh Sumant S 
Published: November 2023

Zinc fingers and homeoboxes (ZHX) transcriptional factor family members, major regulators of podocyte gene expression, are tethered to the cell membrane as heterodimers and homodimers. ZHX2‐ZHX1 heterodimers are mostly present in the podocyte body, and ZHX2‐ZHX3 at the slit diaphragm. Loss of heterodimerization, is common in podocyte diseases and promotes nuclear entry of ZHX proteins.

Human kidney biopsies from focal glomerular sclerosis (FSGS) or minimal change disease (MCD) patients were stained for ZHX proteins. Overall ZHX2 protein expression in FSGS was either increased or decreased, often with major redistribution of ZHX2 and/or ZHX3 to the podocyte nucleus. When studied biopsies from MCD patients, overall expression of ZHX2 and ZHX1 was decreased.

To study the effect of low ZHX2 expression in FSGS, Balb/cJ mice (low ZHX2 content) were treated with Adryamicin (ADR). At baseline, Balb/cJ mice has lower albuminuria levels compared with Balb/c (normal ZHX2 content) but developed higher albuminuria levels at day 14 after ADR treatment (873.53 ± 413.46 μg per 18 h) compared with Balb/c (65.64 ± 18.91 μg per 18 h) (p<0.05)

Podocyte‐specific ZHX2 transgenic rats were generated using the human NPHS2 promoter to study loss of heterodimerization due to ZHX2 overexpression. Three founder lines of ZHX2 podocyte‐specific transgenic rats were characterized (TG 14, TG 142 and TG 144). Glomerular mRNA expression of ZHX2 in heterozygous transgenic rats showed an increase, compared to WT littermates, of 1.13 ± 0.10 in TG 14, 1.50 ± 0.09 in TG 142 and 4.09 ± 0.69 in TG 144.

Baseline confocal characterization of glomeruli in heterozygous TG 144 rats revealed increased expression of ZHX2 in a podocyte cell membrane distribution. Overall expression of ZHX3 and ZHX1 was unchanged. None of the ZHX2 transgenic rat lines had proteinuria at baseline.

When compared with WT littermates, proteinuria and renal histology on Day 7 of ADR was most severe in TG 144 (310.4 ± 41.5 mg per 18 h; p<0.01) and was associated with higher mortality, and also more severe in TG 142 (204.8 ± 29.95 mg per 18 h; p<0.01) than control (102.6 ± 19.5 mg per 18 h) or TG 14 (89.1 ±31.95 mg per 18 h; indistinguishable from control).

ZHX2 transgenic rats were backcrossed for 10 generations into the Buffalo/Mna background, another model of FSGS, showing more proteinuria at age 8 months (301.9 ± 27.4 mg per 18 h) than the Buff/Mna (193.8 ±23.7 mg per 18 h) (p<0.05).

To study the effect of ZHX2 overexpression in MCD, puromycin aminoglucoside (PAN) model was induced in podocye‐specific TG rats. Proteinuria in PAN was less severe in TG rats 10 days after treatment (TG 144 and TG 142 < WT; proteinuria/18h 178.9 ± 13.4 mg, 169.1 ± 37.8 mg and 351.7 ± 38.9 mg, respectively) (p<0.05).

Loss of heterodimerization caused by overexpression or deficiency of ZHX2 in podocytes worsens the development of FSGS, by contrast, overexpression of ZHX2 prevents MCD. These findings suggest a major role of ZHX2 in nephrotic syndrome.

Support or Funding Information

US National Institutes of Health grants (R01DK101637, R01DK109713, R01DK111102, K01DK096127) and American Heart Association grant (16SDG27500017)