Fig 2: 3D culture of kidney organoids. (A) Bright field observations of the kidney organoids. (B) Hematoxylin and eosin staining results of the kidney organoids. (C) Expression detection of renal tubular markers LTL and ECAD in kidney organoids by immunofluorescence. (D) Expression detection of collection tube markers GATA3 and ECAD in kidney organoids by immunofluorescence. Scale bars, 100 µm. (E) Expression detection of kidney podocytes marker PODXL in kidney organoids by immunofluorescence. Scale bars, 50 µm. (F) Expression detection of the molecular markers of podocyte (NPHS1, PODXL and WT1) by western blotting. (G) Expression detection of the specific markers for ureteric buds RET and WNT11 by western blotting. The data are representative from a minimum of three independent experiments. LTL, Lotus tetragonolobus lectin; ECAD, e-cadherin; GATA3, GATA binding protein 3; NPHS1, nephrin; PODXL, podocalyxin; WT1, Wilms tumor protein; RET, proto-oncogene tyrosine-protein kinase receptor Ret; WNT11, protein Wnt-11; D, day.

Fig 3: Family pedigree, mutation analysis and nephrin immunostaining.(A) Pedigree depicting the proband (P, solid arrow) as a compound heterozygote for inherited A419T and C623F mutations in NPHS1. The parents are obligate carriers of each mutation, and the sibling is mutation negative. (B) Functional consequences of A419T and C623F were assessed using protein structure prediction algorithms SIFT (Sorting Intolerant From Tolerant) and PolyPhen (Polymorphism Phenotyping). (C) Schematic representation of nephrin protein and location of mutations under investigation. (D) Immunoperoxidase staining for nephrin in renal biopsies taken from normal control 16-year-old, and the proband at 16 years of age. Compared to the normal control, the proband’s biopsy shows incomplete staining of nephrin along the GBM, with positive staining for nephrin within the podocyte cell bodies. D: original magnification x600. CNS, congenital nephrotic syndrome; IgAN, IgA nephropathy.

Fig 4: PKM2 deficiency in cultured E11 podocytes alleviates LPS-induced nephrin loss. A Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of PKM2, PKM1, and PKM1/2 levels in undifferentiated (day 1; D1) and differentiated (D15) E11 murine podocytes infected with lentivirus particles carrying scramble-shRNA (SCR), shRNA targeting PKM2 (M2KD) or an open reading frame of the human DNA (M2R). ß-Actin was used as a loading control. *p < 0.05, **p < 0.01 indicate a significant difference between differentiated and undifferentiated cells. #p < 0.05, ##p < 0.01 indicate a significant difference between the indicated cell and control E11 cells exposed to the same treatment. B Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of pPKM2Y105, pPKM2S37, PKM2, PKM1, PKM1/2, and Nephrin in differentiated M2R and M2KD podocytes treated with LPS for the indicated durations. ß-Actin was used as a loading control. Data is representative of at least three independent experiments. *p < 0.05, **p < 0.01 indicate a significant difference between LPS-treated or non-treated cells. &p < 0.05, &&p < 0.01 indicate a significant difference between M2R and M2KD cells exposed to the same treatment

Fig 5: PKM2 podocyte deletion ameliorates LPS induced proteinuria and kidney injury. A Representative immunoblots (left panel) of PKM2, PKM1, PKM1/2, and ß-Actin in whole kidney lysates from wild type (Ctrl) and podocyte PKM2 knockout mice (KO). mRNA levels (right panel) of Pkm2 in total kidney lysates of Ctrl and KO mice (n = 6 group). B Representative immunoblots (left panel) of PKM2, PKM1, PKM1/2, and ß-Actin in primary podocytes isolated from Ctrl and KO mice. The right panel is the mRNA level of Pkm2 and Pkm1 C in primary podocytes (n = 12; 4 animals/lane). D Body weights, E kidney weights, and F kidney to body weight ratio of wild type (Ctrl) and podocyte PKM2 knockout mice (KO) mice, under PBS and LPS treated states. Assessment of G total urinary proteins levels, H serum albumin levels, I urine albumin levels, J albumin to creatinine ratios (ACR), and K blood urea nitrogen (BUN) levels of Ctrl and KO mice after PBS and LPS injection (n = 6–12 per group). L PAS staining of kidney sections in Ctrl and KO mice under PBS and LPS conditions; the arrow is pointing to the expansion of Bowman space in response to LPS treatment. Scale bar = 50 µM. M Immunofluorescence of Nephrin (green), PKM2 (red), and nuclear DNA using DAPI (blue) in kidney sections obtained from both genotypes injected with PBS or LPS. Scale bar = 50 µM. In D–K, *p < 0.05, **p < 0.01 indicate a significant difference between PBS- and LPS-injected mice. &p < 0.05, &&p < 0.01 indicate a significant difference between Ctrl and KO mice