Overview
Wolf Lab is interested in the regulation of ion channels in the kidney, specifically in calcium and magnesium channels and their impact on metabolism. Our focus is on urinary proteins such as Uromodulin, the most abundant protein in human urine, and Mucin-1 as regulators of renal calcium and magnesium absorption. We investigate how they affect tubular calcium and magnesium homeostasis and thereby modify the risk of kidney stones or diabetes mellitus in mouse models and humans. Mutations in Uromodulin cause a human kidney disease called “Autosomal Dominant Tubulo-Interstitial Kidney Disease” (ADTKD) and we want to identify new avenues for the treatment of ADTKD.
Our projects involve different lines of experimental approaches and usually start with electrophysiological studies that allow us to develop a hypothesis of a signaling pathway. We then apply protein biochemistry to test this hypothesis and to better understand protein interactions and expression. In a final step, we use in vivo models to confirm our hypothesis in mouse models or humans. Our experimental spectrum includes different forms of patch-clamp studies (whole-cell, single channel, primary cells in split open tubules), advanced molecular biology, protein biochemistry, high-throughput assay screening, tissue culture, creation of stably transfected cell lines, in vivo work with genetically modified mice, and translational studies with human patients.
Research
Urinary proteins such as Uromodulin and Mucin-1 modify tubular calcium absorption and are reduced in kidney stone formers
About 50% of all kidney stones contain calcium. We found that patients with calcium-containing kidney stones had significantly less Mucin-1 in the urine compared to control individuals. When investigating the renal expression of Mucin-1 we found Mucin-1 expression along the thick ascending limb, the distal convoluted tubule, and the collecting duct. We identified that Mucin-1 stimulates the renal calcium channel TRPV5 from the extracellular side (e.g. from the ultrafiltrate) by interacting with the N-glycan of TRPV5. Mucin-1 polymerizes in the ultrafiltrate and we hypothesize that it forms a lattice. Our data are consistent with Mucin-1 lattice enhancing tubular calcium absorption by interfering with the endocytosis of TRPV5, thereby reducing the supersaturation of calcium and risk for calcium-containing kidney stones. A ubiquitous urinary lectin called galectin-3 is required for the Mucin-1 mediated stimulation of TRPV5. We are now interested to further dissect the mechanism of how galectins are involved in this process. We want to investigate if stone formers secrete urinary Uromodulin and Mucin-1 in a similar fashion as control individuals when environmentally challenged.
Mucin-1 (Muc1) is expressed in different nephron segments. A-C' Muc1 immunostaining in mouse kidney.(A) shows tubules at low magnification. (A') shows higher magnification of (A) with Muc1 localizing to medullary tubules containing basolateral Na+/K+ ATPase (red) which depicts thick ascending limb. (B, B') Muc1 (green) localizes to tubules with NCC (red, a marker of DCTs) but not with Lotus tetragonolobus (LTL) (blue, marker of PTs) and (C, C') to tubules with Aquaporin 2 (AQ2) (red, a marker of the collecting ducts). Pictures were obtained in cooperation with the Marciano Lab at UTSW. Uromodulin enhances tubular magnesium absorption and modifies the risk for type 2 diabetes mellitus.
Uromodulin enhances tubular magnesium absorption and modifies the risk for type 2 diabetes mellitus.
In humans, magnesium supplementation improves diabetic control whereas hypomagnesemia induces diabetes mellitus. Patients with Gitelman syndrome, a magnesium-wasting tubulopathy, or patients taking calcineurin inhibitors, which causes hypomagnesemia by urinary magnesium wasting, have a higher risk for diabetes mellitus. In hypomagnesemic mice, mRNA expression of the urinary protein Uromodulin is upregulated suggesting a role of Uromodulin in renal magnesium absorption. Uromodulin is secreted into the ultrafiltrate in the thick ascending limb (TAL) and to a lesser degree in the distal convoluted tubule (DCT).
We developed a model where Uromodulin polymerizes in the urine and forms a lattice with the N-glycan of the renal magnesium channel TRPM6 in the DCT. The ubiquitous urinary lectin Galectin-1 is required for the enhancement of magnesium absorption via TRPM6, possibly by stabilizing the lattice. The lattice impairs the retrieval of TRPM6 channels from the surface and allows thereby for more magnesium absorption. Interestingly, in a genome-wide association study, Mucin-1 was also associated with hypomagnesemia. We are now investigating the effect of Mucin-1 on renal magnesium homeostasis. We are studying if the enhanced magnesium absorption of magnesium by Uromodulin and Mucin-1 have an effect on metabolism and glucose tolerance.
Mechanistic model how Uromodulin increases abundance of TRPM6 in the distal convoluted tubule. Small molecules enhance secretion of mutant Uromodulin
Uromodulin mutations lead to autosomal dominant tubulo-interstitial kidney disease (ADTKD) which is the most common inherited kidney disease after autosomal dominant polycystic kidney disease, and Collagen IV mutations. So far no treatment is available for patients with Uromodulin mutations. The disease often manifests at the age 10-20 years and leads to kidney failure at the age of 30-50 years. Due to the mutations found in Uromodulin, the mutant protein is not secreted into the urine and accumulates (MUT vs. WT) within the tubular cells. This process leads to apoptosis over time. Patients with Uromodulin mutations have significantly less Uromodulin in the urine (lanes 1 and 4 in the western-blot of human urine are ADTKD are patients, lanes 2 and 3 are control individuals). We have developed a cell culture based assay that allows us to monitor secretion of wild-type and mutant Uromodulin. In a proof-of-concept study, we screened 8,000s compounds with the High-Throughput Screening Core at UTSW and identified 10 promising compounds that enhanced mutant Uromodulin secretion almost to the level of wild-type UMOD. We are now planning to screen the larger 300,000 compound containing library to identify potentially new therapies for ADTKD caused by Uromodulin mutations.
Whereas wild-type (WT) Uromodulin localizes mostly to cell membranes and the cytosol the mutant Uromodulin (MUT) barely localizes to cell membranes and forms intracellular aggregates. Urine of patients with Uromodulin mutations contains very little Uromodulin (lanes 1 and 4 of the SDS gel) whereas control individuals have abundant urinary Uromodulin (lanes 2 and 3 of the SDS gel). Focus of the Wolf Lab
- Study the effects of urinary Uromodulin and Mucin-1 on renal calcium absorption and the risk of kidney stone formation
- Investigate the effects of urinary Uromodulin and Mucin-1 on tubular magnesium absorption and metabolism
- Identify new targets for the treatment of ADTKD caused by Uromodulin mutations using a high-throughput assay