Shabbir H. Moochhala

Renal calcium stones: insights from the control of bone mineralization

Extracellular pyrophosphate (PPi) plays a central role in the control of normal bone mineralization since it antagonizes inorganic phosphate in the promotion of hydroxyapatite deposition. Studies using knock‐out mice have established the functional importance of PPi generation via nucleotide pyrophosphatase phosphodiesterases (NPP) and of PPi transmembrane transport by the progressive ankylosis (ANK) protein. Tissue non‐specific alkaline phosphatase activity counteracts this by hydrolysis of PPi to inorganic phosphate. The molecular nature and transport function of ANK are reviewed. A close parallel is drawn between the controlled mineralization of bone and the prevention of abnormal calcium crystal deposition within the kidney, especially when concentrated urine is produced. Pyrophosphate is present in urine, and ANK is expressed in the cortical collecting duct where PPi transport to both the tubular lumen and the renal interstitium may occur. Pyrophosphate may also be generated here by nucleoside triphosphate diphosphohydrolases (NTPD2 and 3) together with NPP1. Alkaline phosphatase activity is restricted to the proximal nephron, remote from these sites of PPi generation, transport and function. The physiological importance of PPi generation and transport in preventing idiopathic calcium renal stone disease and nephrocalcinosis now needs to be established.

The Medical Management of Urolithiasis

Urolithiasis is increasing in prevalence and causes substantial morbidity and economic burden. Aside from “acute” stone episodes, urolithiasis should be regarded as a systemic disorder and investigated fully. Practical guidance is given for the medical investigation, diagnosis and treatment of urolithiasis. A single episode of urolithiasis should prompt a review of potential risk factors for recurrent disease and appropriate baseline investigations should be performed. Further biochemical tests may then be directed appropriately and allow individualised advice and treatment to be given to the patient with urolithiasis.

The pyrophosphate transporter ANKH is expressed in kidney and bone cells and colocalises to the primary cilium/basal body complex.

Background/Aims: ANKH encodes a putative pyrophosphate transporter named ANKH, which regulates tissue calcification. ANKH is a transmembrane protein with at least 8 predicted transmembrane domains. Sequence analysis reveals a possible cilial localisation motif immediately after the last transmembrane segment. Here we aim to determine the subcellular localisation of ANKH in ciliated epithelial cells and murine tissue and identify colocalisation using ciliary/basal body markers. Methods: Using murine kidney, renal epithelial cells and osteoblast cells we investigated the expression and localisation of ANKH using RT-PCR, Western blotting and immunocytochemistry. Results: Here we confirm endogenous expression of ANKH mRNA and protein in whole mouse kidney as well as mouse renal epithelial cell lines M1 and mpkCCDcl4 and the osteoblast cell line MC3T3-E1. Using antibodies directed towards ANKH, we confirm cilial and basal body localisation in renal tissues and renal epithelial cells, in addition to a centrosomal localisation in dividing mpkCCDcl4 cells. We also establish that the osteoblast cell line MC3T3-E1 forms an epithelioid cell layer, with junctional complex formation and primary cilia expression. ANKH is also seen within cilial and basal body structures of MC3T3-E1 cells. An ANKH-3XFLAG construct expressed in mpkCCDcl4 cells also localises to the primary cilium/basal body complex confirming this localisation. Conclusion: We conclude that the transmembrane protein ANKH is expressed in cilia and basal body structures, and postulate a sensory role at this location.

Nephrocystin-1 interacts directly with Ack1 and is expressed in human collecting duct

Nephronophthisis is characterised by renal fibrosis, tubular basement membrane disruption and corticomedullary cyst formation leading to end stage renal failure. Mutations in NPHP1 account for the underlying genetic defect in 25% of patients with nephronophthisis. Loss of urine concentration ability may be an early feature of nephronophthisis. Using yeast-2-library screening with the SH3 domain of nephrocystin-1 as bait, we identify Ack1 as a novel interaction partner. This interaction is confirmed using exogenous over-expression followed by co-immunoprecipitation. Ack1 is an activated Cdc42-associated kinase, and like nephrocystin-1, is a known interactor of p130Cas. Nephrocystin-1 partially colocalises with Ack1 at cell-cell contacts in IMCD3 cells. In human kidney, nephrocystin-1 expression is limited to cell-cell junctions in renal collecting duct cells. These data define Ack1 as a novel interaction partner of nephrocystin-1 and implicate cell-cell junctions and the renal collecting duct in the pathology of nephronophthisis.

Identification of compound heterozygous KCNJ1 mutations (encoding ROMK) in a kindred with Bartter's syndrome and a functional analysis of their pathogenicity

A multiplex family was identified with biochemical and clinical features suggestive of Bartters syndrome (BS). The eldest sibling presented with developmental delay and rickets at 4 years of age with evidence of hypercalciuria and hypokalemia. The second sibling presented at 1 year of age with urinary tract infections, polyuria, and polydipsia. The third child was born after a premature delivery with a history of polyhydramnios and neonatal hypocalcemia. Following corrective treatment she also developed hypercalciuria and a hypokalemic metabolic alkalosis. There was evidence of secondary hyperreninemia and hyperaldosteronism in all three siblings consistent with BS. Known BS genes were screened and functional assays of ROMK (alias KCNJ1, Kir1.1) were carried out in Xenopus oocytes. We detected compound heterozygous missense changes in KCNJ1, encoding the potassium channel ROMK. The S219R/L220F mutation was segregated from father and mother, respectively. In silico modeling of the missense mutations suggested deleterious changes. Studies in Xenopus oocytes revealed that both S219R and L220F had a deleterious effect on ROMK‐mediated potassium currents. Coinjection to mimic the compound heterozygosity produced a synergistic decrease in channel function and revealed a loss of PKA‐dependent stabilization of PIP2 binding. In conclusion, in a multiplex family with BS, we identified compound heterozygous mutations in KCNJ1. Functional studies of ROMK confirmed the pathogenicity of these mutations and defined the mechanism of channel dysfunction.

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy as a bedside diagnostic tool for detecting renal disease biomarkers in fresh urine samples

Attenuated total reflection (ATR)-FTIR spectroscopy is a convenient technique for analysing biomedical samples because of its sensitivity to subtle compositional changes, speed of data acquisition and ease of sample preparation. We have applied the technology to the detection of disease biomarkers in urine and investigated the translation of these diagnostic methods to simple bench-top spectrometers. To demonstrate the use of ATR-FTIR spectroscopy as a bedside diagnostic tool, we have installed a roomtemperature bench-top infrared spectrometer in the renal unit at the Royal Free Hospital (RFH), London. A nurse recorded spectra of urine from patients with a range of conditions, including diabetes, kidney disease, stone disease and urinary tract infections, and the data were correlated to medical conditions to assess the diagnostic capabilities of the system and to identify potential spectral patterns associated with disease. Two hundred and six spectra have been recorded to date; these show it is possible to detect urea, creatinine, protein, lipids, sugars and other minor metabolites, including potential disease biomarkers. Several spectral peaks of potential diagnostic interest were identified that show variations between normal and disease samples.

Infrared vibrational spectroscopy: a rapid and novel diagnostic and monitoring tool for cystinuria

Cystinuria is the commonest inherited cause of nephrolithiasis (~1% in adults; ~6% in children) and is the result of impaired cystine reabsorption in the renal proximal tubule. Cystine is poorly soluble in urine with a solubility of ~1 mM and can readily form microcrystals that lead to cystine stone formation, especially at low urine pH. Diagnosis of cystinuria is made typically by ion-exchange chromatography (IEC) detection and quantitation, which is slow, laboursome and costly. More rapid and frequent monitoring of urinary cystine concentration would significantly improve the diagnosis and clinical management of cystinuria. We used attenuated total reflection - Fourier transform infrared spectroscopy (ATR-FTIR) to detect and quantitate insoluble cystine in 22 cystinuric and 5 healthy control urine samples. Creatinine concentration was also determined by ATR-FTIR to adjust for urinary concentration/dilution. Urine was centrifuged, the insoluble fraction re-suspended in 5 μL water and dried on the ATR prism. Cystine was quantitated using its 1296 cm−1 absorption band and levels matched with parallel measurements made using IEC. ATR-FTIR afforded a rapid and inexpensive method of detecting and quantitating insoluble urinary cystine. This proof-of-concept study provides a basis for developing a high-throughput, cost-effective diagnostic method for cystinuria, and for point-of-care clinical monitoring

Pyrophosphate Transport and Stones

Since the 1960’s, inorganic pyrophosphate (PPi) has been known to inhibit apatite precipitation. Recent findings suggest that PPi plays a central role in the control of normal bone mineralization. Knockout mice have established the functional importance of PPi transmembrane transport, via the pyrophosphate transporter ANKH. The molecular nature and transport function of ANKH are reviewed. PPi is present in urine and ANKH is expressed in the cortical collecting duct where PPi transport to both the tubular lumen and renal interstitium may occur. Arginine vasopressin stimulation of cortical collecting duct cells grown on semi‐permeable supports appears to upregulate apical ANKH expression, which we postulate may be a mechanism of stone inhibition during urinary concentration and supersaturation of calcium salts. Hypopyrophosphaturia may be a forgotten metabolic risk factor for stone formation and polymorphisms of the ANKH gene may underlie this defect. The physiological importance and clinical significance o...