Figure 6.

Uromodulin (UMOD) investigations. (a) Plasma concentration of UMOD decreases with reduced estimated glomerular filtration rate (ml/min per 1.73 m²) in individuals affected with the missense variant p.R79W in ALG5, individuals with UMOD pathogenic variants and individuals with unspecified CKD. The decrease in UMOD concentration is slower and less expressive in cases with the ALG5 variant than in cases with UMOD mutations. A best-fit line is included for each group (b) Semiquantitative and qualitative detection of urinary UMOD. Western blot of spot urine samples from 10 healthy controls, 1 patient with a canonical variant in UMOD (ADTKD-UMOD), and 6 genetically affected individuals from family F350 and family F200 showed a reduced amount of urinary UMOD in all investigated patients compared to healthy controls. No abnormality in electrophoretic mobility of residual UMOD suggestive of abnormal N-linked glycosylation was observed in patients. The sample volume was normalized to urinary creatinine level. Intracellular localization of UMOD in patients and control kidney biopsy. To elucidate the cause of the reduction of urinary excretion and the plasma level of UMOD, we analyzed its intracellular localization in kidney biopsy from the patient and control. In affected kidney biopsy, parallel staining of uromodulin (UMOD) with Protein disulfide isomerase (PDI), a marker of endoplasmic reticulum (ER), and with pan-Cadherin, (c in detail d a marker of the plasma membrane (PM)) demonstrate localization of the uromodulin in both, (d) partly in the ER (34%) and (f) partly on PM (66%). In the control kidney, (g in detail h) parallel staining of UMOD with PDI and pan-Cadherin demonstrated (i) localization of UMOD on PM (92%) (j) but not in the ER. In the upper right corner of each image (6K, 6F, 6I, 6J) is the Pearson coefficient ± SEM to show mean degree of colocalization between individual compartment markers and ALG5 in all analyzed tubules. The degree of UMOD colocalization with selected markers is demonstrated by the fluorescent signal overlap coefficient values ranging from 0 to 1. The resulting overlap coefficient values are presented as the pseudo-color, which scale is shown in the corresponding lookup table.