Several studies have shown that calcium phosphate (CaP) stones are formed in the early segments of the nephron, namely the proximal tubule (PT) and the loop of Henle (LOH), where conditions are favorable due to high calcium (Ca2+) and phosphate concentrations, as well as a relatively high pH. The PT is the major site for Ca2+ reabsorption, where a paracellular pathway has been reported. However, existence of any regulated Ca2+ transport through a transcellular route is unknown. Our present proposal will study this yet unknown regulated Ca2+ entry mechanism that controls transcellular Ca2+ transport, which has a role in stone formation. Our preliminary data show that Ca2+-sensing receptor (CSR), a G protein-coupled receptor that responds to alterations in extracellular [Ca2+] ([Ca2+]o), and a transient receptor potential canonical 3 (TRPC3), a Ca2+ permeable channel, both localize at the luminal region of PT cells. Our data show also that: 1) CSR couples with TRPC3 both physically and functionally; and 2) [Ca2+]o mediates this coupling response through CSR which signals TRPC3 channels via a phospholipase C (PLC)-dependent pathway. More importantly, we found that the pharmacological/genetic disruption of both CSR and TRPC3 markedly attenuated this Ca2+ influx in PT cells and that TRPC3-null mice displayed a phenotype of elevated [Ca2+] in urine, calcification in kidney and scattered crystals in the urine and the LOH. Based on our preliminary data, we hypothesize that increased [Ca2+] and other modulators, like protons and amino acids, in PT luminal fluid can activate CSR-TRPC3 signaling via a PLC-dependent pathway, thereby initiating transcellular Ca2+ transport across the PT. We further hypothesize that such a mechanism to increase Ca2+ transport plus the acidification of the PT luminal fluid together serves to prevent the nucleation of CaP stone at the LOH. We have the following specific aims to test this hypothesis.
Aim 1 proposes to determine the mechanism of CSR-mediated Ca2+ entry/transport into PT cells by determining the role of CSR-TRPC3 signaling in Ca2+ entry/transport in PT cells using TRPC3 knockout (KO) mice and the pharmacological/genetic disruption of CSR-TRPC3 signaling.
In Aim 2, we propose to study the Ca2+ entry/transport in vivo in TRPC3 KO mice, and to disrupt the phosphate and oxalate transport mechanism in TRPC3 KO mice by introducing in vivo siRNA application to PT to favor the process of CaP and CaP+CaOx stone formation at LOH. Finally, in Aim 3, we plan to rescue the phenotype (e.g., normalize [Ca2+] in urine) of TRPC3 KO mice and determine the role of increased [Ca2+] and pH in PT and its contribution to CaP stone formation by acidifying or alkalinizing the urine with or without inducing hypercalcemia in TRPC3 KO mice, and then measure the urine properties and degree of calcification/stone formation in LOH. Proposed aims will unravel novel mechanisms: i) the regulated transcellular Ca2+ transport in PT; and ii) maintenance of [Ca2+] in PT luminal fluid. Information gained will help to understand the formation of CaP stone that could potentially lead to the development of new therapeutic strategies.
The prevalence of kidney stones in the United States has risen over the past 30 years. Diagnosis, treatment, high recurrence, hospitalizations, surgery, and lost work time due to these stones cost billions of dollars yearly in the United States. Stone formation is also associated with increased rates of chronic kidney disease and hypertension which are not completely explained by obesity, a risk factor in each of these conditions. Additionally, by 70 years of age, 11% of men and 5.6% of women will develop a stone. The majority (80%) of these calcium stones is found to be calcium oxalate (CaOx), with variable amounts of calcium phosphate (CaP). Research conducted over the past 3-4 decades focused only on delineating the mechanism of CaOx stone formation. However, over the past two decades, there is a documented increase in the prevalence of pure CaP stones in the kidney, suggesting the need for research about the formation of CaP stones to develop new clinical interventions and therapy. The proposed research will thus provide the evidence for the molecular candidates that governs the regulation of calcium concentration inside the proximal tubular lumen to focus on the mechanism of formation of these CaP stones, as well as the potential in vivo interventions in our animal model. Additionally, the proposed study will shed light on the aggregation of CaP crystals (amorphous) found in Randall's plaque, which is created from large spherulites of CaOx that grows over this CaP nidus. Therefore information gained from the proposed study can potentially lead to the development of new therapeutic strategies for both CaP and CaOx stone formation.
| Lau, Ivan; Potluri, Ajay; Ibeh, Cliff-Lawrence et al. (2017) Microcalcifications in stone-obstructed human submandibular gland are associated with apoptosis and cell proliferation. Arch Oral Biol 82:99-108 |
| Yiu, Allen J; Ibeh, Cliff-Lawrence; Roy, Sanjit K et al. (2017) Melamine induces Ca2+-sensing receptor activation and elicits apoptosis in proximal tubular cells. Am J Physiol Cell Physiol 313:C27-C41 |
