Harris, Peter C

Stem cell-derived kidney organoids enable human genetic disease modeling to identify the parathyroid hormone 1 receptor as a potential new therapeutic target for developmental polycystic kidney disease.

ABSTRACT Background Primary hyperoxaluria (PH), a rare autosomal recessive disease of oxalate accumulation in the kidneys, is caused by biallelic pathogenic changes in three known genes: AGXT (PH1), GRHPR (PH2) and HOGA1 (PH3). Methods To better understand the overall risk of developing clinical PH, we manually curated and classified PH genetic variants and calculated the estimated genetic prevalence overall and in five ethnic subpopulations using allelic frequencies from the population Genome Aggregation Database (gnomAD version 2.1.1). Results Of the 651 identified PH variants, 273 were found in gnomAD 2.1.1 on the day of download and after reclassification, 208 were determined pathogenic (P) or likely pathogenic (LP) (AGXT, n = 94; GRHPR, n = 46; and HOGA1, n = 68) and a further 65 were classified as rare variants of uncertain significance (VUS). Using P and LP only, estimated carrier frequency was 1:229 for PH1, 1:465 for PH2 and 1:151 for PH3, while genetic prevalence was 1:209 357 for PH1, 1:863 028 for PH2 and 1:90 834 for PH3 (i.e. nearly 5, 1 and 11 per 1 million individuals, respectively). The highest carrier frequencies for AGXT pathogenic variants were in East Asians (1 in 146) and the European non-Finnish population (1 in 187); for GRHPR, South Asians (1 in 313) and the European non-Finnish population (1 in 413); and for HOGA1, Ashkenazi Jewish (1 in 38) and East Asians (1 in 100). The estimated risk of developing PH was ≈1:59 017. Conclusions This careful benchmarking exercise indicates that a significant number of individuals at risk for PH symptoms remain undiagnosed. Since these numbers exceed known diagnosed cases of PH, improved screening and diagnosis of this underestimated disease is necessary.

Key Points Circulating proteins independently associated with kidney stone diagnosis are related to kidney stone matrix. Lower circulating uromodulin and higher scavenger receptor cysteine-rich domain-containing group B protein levels were associated with higher kidney stone risk, whereas kidney stone presence elevates plasma matrix metalloproteinase 7 level. The protective effect of uromodulin against kidney stones was independent of kidney function. Background Kidney stones are increasingly recognized as a systemic disorder with a high global prevalence. However, large proteomics studies are lacking. Methods An individual-level proteomics study with rigorous adjustments was performed on 35,331 UK Biobank participants to uncover the independent associations between 2922 circulating proteins and prevalent kidney stone disease. Mendelian randomization analysis was used to assess causal relationships. Findings were validated using genomic data from the Mayo Clinic Biobank ( N =43,744), concentration-response analysis, transcriptomics analyses, and additional genome-wide association studies. Results Nine plasma proteins were independently associated with a kidney stone diagnosis, including reduced uromodulin (UMOD; β , −0.10; 95% confidence interval [CI], −0.15 to −0.05) and elevated scavenger receptor cysteine-rich domain-containing group B protein (SSC4D; β , 0.28; 95% CI, 0.17 to 0.38) and were enriched in extracellular matrix pathways. Mendelian randomization analysis revealed that the presence of kidney stone contributed to elevated levels of matrix metalloproteinase 7. Conversely, lower plasma UMOD (odds ratio [OR], 0.93; 95% CI, 0.90 to 0.97) and higher plasma SSC4D (OR, 1.10; 95% CI, 1.02 to 1.18) were associated with kidney stone risk. These associations were consistently replicated in the Mayo Clinic Biobank dataset (UMOD; OR, 0.92; 95% CI, 0.86 to 0.98; SSC4D; OR, 1.13; 95% CI, 1.01 to 1.27) and further validated by a concentration-response analysis. Single-nucleus RNA sequencing and quantitative trait loci analyses confirmed consistent associations between thick ascending limb UMOD expression and stone former status and with blood and urine UMOD concentrations. Genome-wide association study analysis, adjusted for eGFR, suggested that the protective role of UMOD against kidney stones was independent of kidney function. Conclusions This study highlights significant associations between concentrations of specific blood proteins and a history of kidney stones. Several implicated proteins are related to kidney stone matrix, with UMOD independently associated with lower risk and SSC4D with higher risk of kidney stones.

ABSTRACT Human alanine:glyoxylate aminotransferase (AGT) is a liver peroxisomal enzyme that metabolizes glyoxylate, the oxalate precursor, to glycine. AGT deficiency, due to recessive pathogenic changes in the AGXT gene, results in calcium oxalate accumulation and kidney stones, a condition known as primary hyperoxaluria type 1 (PH1). Most missense variants lead to PH1 by causing AGT misfolding, but their effects manifest differently based on the presence of two polymorphic variants, p.Pro11Leu (p.P11L) and p.Ile340Met (p.I340M), which are usually present in linkage disequilibrium and generate the minor haplotype. While the p.P11L substitution reduces AGT global stability and worsens the effects of pathogenic changes, the p.I340M exerts a stabilizing effect whose role on PH1 pathogenesis has never been elucidated. The p.Ile279Thr (p.I279T) variant is frequent in healthy populations (0.29%), mainly on the major allele, but we present data from six PH1 families (eight patients) suggesting p.I279T as a PH1 pathogenic allele. Interestingly, in these families, p.Thr279 is always associated with p.Leu11 and p.Ile340, thus with a split AGXT haplotype. Analysis of the effects of the p.I279T mutation by in silico predictions, biochemical analyses on purified proteins, and expression in two cellular models of disease ( AGXT1 ‐KO HepG2 and CHO cells) shows that it causes a folding defect that is exacerbated by p.P11L but mitigated by p.I340M, thus explaining why p.Thr279 is pathogenic just on the p.Leu11–p.Ile340 haplotype. These data indicate that genetic screenings for PH1 should document the AGXT haplotype, including its components, to obtain an accurate diagnosis and possible prognostic information.

Key Points Loss-of-function ALG8 and ALG9 variants were enriched in polycystic kidney/liver groups and International Classification of Diseases–coded cystic individuals in population cohorts. The ALG8 and ALG9 kidney phenotypes were usually mild to moderate, and lower eGFR or kidney failure was rare. ALG8 pathogenic variants sometimes resulted in severe polycystic liver disease. Background Autosomal dominant polycystic kidney disease (ADPKD) is a common, inherited nephropathy often resulting in kidney failure. It is genetically heterogeneous; along with the major genes, PKD1 and PKD2 , at least eight others have been suggested. ALG8 pathogenic variants have been associated with autosomal dominant polycystic liver disease and implicated in ADPKD, while ALG9 has been suggested as an ADPKD gene, but details of the phenotypes and penetrance are unclear. Methods We screened >3900 families with cystic kidneys and/or livers using global approaches to detect ALG8 or ALG9 pathogenic variants. In addition, population cohorts with sequence data (Genomics England 100K Genomics Project, UK Biobank, and Mayo Clinic Biobank [MCBB]) were screened for ALG8 / ALG9 pathogenic variants. Results Multicenter screening of individuals with polycystic kidney and/or liver disease identified 51 (1.3%) ALG8 (7 multiplex) and 23 (0.6%) ALG9 (5 multiplex) families—frequencies that were approximately 10× and approximately 24× greater than nonpolycystic kidney disease controls. Analysis of individuals with polycystic kidney disease phenotypes in 100K Genomics Project, UK Biobank, and MCBB identified nine ALG8 (0.39%) and nine ALG9 (0.39%) families, an enriched frequency over controls. Two individuals had PKD1 and ALG8 pathogenic changes. Eighty-nine percent of individuals with ALG8 mutations with imaging in the entire MCBB had kidney cysts (50%, >10 cysts), with greater median kidney and liver cyst numbers than controls. For ALG9, 78% had kidney cysts (27%, >10 cysts). Individuals with ALG8 mutations typically had mild cystic kidneys with limited enlargement. Liver cysts were common (71%), with enlarged livers (>2L) found in 11 of 62 patients, although surgical intervention was rare. The ALG9 kidney phenotype was also of mild cystic kidneys, but enlarged livers were rare; for both genes, CKD or kidney failure were rare. Conclusions ALG8 and ALG9 are defined as cystic kidney/liver genes but with limited penetrance for lower eGFR.

Key Points PIEZO1 activation reduces cystogenesis: Yoda1 activates PIEZO1, raising calcium and lowering cAMP, reducing cyst growth in autosomal dominant polycystic kidney disease models. Context-dependent role of PIEZO1: PIEZO1 knockout in mice with or without polycystic kidneys does not affect cyst formation, suggesting redundancy in mechanosensitive pathways. Therapeutic potential: Findings support PIEZO1 activation as a part of combination therapy to slow cyst growth in autosomal dominant polycystic kidney disease, needing more in vivo studies. Background The disruption of calcium signaling associated with polycystin deficiency is a key factor in abnormal epithelial growth in autosomal dominant polycystic kidney disease. Calcium homeostasis can be influenced by mechanotransduction. The mechanosensitive cation channel PIEZO1 has been implicated in sensing intrarenal pressure and regulating urinary osmoregulation, but its role in kidney cystogenesis is unclear. Methods We hypothesized that altered mechanotransduction contributes to cystogenesis in autosomal dominant polycystic kidney disease and that activation of mechanosensitive cation channels could be a therapeutic strategy. Results We demonstrate that Yoda1, a PIEZO1 activator, increases intracellular calcium and reduces forskolin-induced cAMP levels in mouse inner medullary collecting duct (mIMCD3) cells. Notably, knockout of polycystin-2 attenuated the efficacy of Yoda1 in reducing cAMP levels in mIMCD3 cells. Yoda1 also reduced forskolin-induced mIMCD3 cyst surface area in vitro and cystic index in mouse metanephros ex vivo in a dose-dependent manner. However, collecting duct–specific PIEZO1 knockout neither induced cystogenesis in wild-type mice nor altered cystogenesis in the Pkd1 RC/RC mouse model. Conclusions These findings support the potential role of PIEZO1 agonists in mitigating cystogenesis by increasing intracellular calcium and reducing cAMP levels, but the unaltered in vivo cystic phenotype after PIEZO1 knockout in the collecting duct suggests possible redundancy in mechanotransductive pathways.

Key Points Robotic ultrasound performed favorably compared with magnetic resonance imaging for evaluating total kidney volume. Collagen evaluation by two novel methods of picrosirius red imaging were more informative than the standard method by brightfield imaging. Findings can improve research by increasing speed and access to total kidney volume determination and sensitivity of collagen assessment. Background 3D imaging and histology are critical tools for assessing polycystic kidney disease (PKD) in patients and animal models. Magnetic resonance (MR) imaging provides micron resolution but is time consuming and expensive, and access to equipment and expertise is limited. Robotic ultrasound (US) imaging has lower spatial resolution but is faster, more cost-effective, and accessible. Similarly, picrosirius red (PSR) staining and brightfield microscopy are commonly used to assess fibrosis; however, alternative methods have been shown in non-kidney tissues to provide greater sensitivity and more detailed structural characterization. Methods In this study, we evaluated the utility of robotic US and alternative methods of quantifying PSR staining for PKD research. We compared longitudinal total kidney volume measurements using US and MR imaging. We additionally compared PSR imaging and quantification using standard brightfield microscopy with that by circularly polarized light with hue analysis and fluorescence imaging analyzed using curvelet transform fiber extraction software for automatic detection of individual collagen fibers. Results Increased total kidney volume was detected by US in Pkd1 RC/RC versus wild-type (WT) at time points spanning from early to established disease. US interobserver variability was greater but allowed scanning in 2–5 minutes/mouse, whereas MR imaging required 20–30 minutes/mouse. While no change in fibrotic index was detected in this cohort of relatively mild disease using brightfield microscopy, polarized light showed fibers skewed thinner in Pkd1 RC/RC versus WT. Fluorescence imaging showed a higher density of collagen fibers in Pkd1 RC/RC versus WT, and fibers were thinner and curvier with no change in length. In addition, fiber density was higher in both glomeruli and tubules in Pkd1 RC/RC , and glomeruli had a higher fiber density than tubules in Pkd1 RC/RC and trended higher in WT. Conclusions These studies show robotic US is a rigorous imaging tool for preclinical PKD research. In addition, they demonstrate the increased sensitivity of polarized and fluorescence analysis of PSR-stained collagen.

Key Points Common kidney stones are unlikely to be an independent and direct cause of CKD in the general population. CKD may protect against kidney stones because of changes in key urinary factors critical for stone formation. Background Kidney stones and CKD are common disorders with a substantial interaction. Although observational studies have suggested a potential for enhanced CKD risk after prior kidney stones, the exact relationship remains ambiguous. Methods Shared comorbidities between two diseases were identified using unbiased screening. Genome-wide association study summary statistics were obtained from the UK Biobank (UKBB), FinnGen, and CKDGen, followed by genetic association analyses across various traits. Bidirectional Mendelian randomization (MR) analyses were performed to define causal links, complemented by multivariable MR that included the shared comorbidities including hypertension, diabetes, and obesity. Observational analyses were undertaken using cohorts from the Mayo Clinic and a UKBB subset. Results Despite identifying a total of 123 conditions as shared comorbidities, there was no significant genetic correlation between kidney stones and CKD. Unadjusted MR analysis revealed no significant association between kidney stones and CKD risk (UKBB [exposure]/FinnGen [outcome]: odds ratio [OR]=0.97, 95% confidence interval [CI], 0.88 to 1.06; FinnGen/UKBB: OR=1.17, 95% CI, 0.98 to 1.39). Kidney stones did significantly associate with a higher urinary albumin-creatinine ratio ( β =0.014, 95% CI, 0.002 to –0.025), but this association disappeared in the multivariable MR model ( β =0.009, 95% CI, −0.003 to 0.020). Furthermore, in a cross-sectional analysis limited to the UKBB cohort, a robust regression model did not detect an independent association between kidney stones and urinary albumin-creatinine ratio ( β =0.16, 95% CI, −0.04 to 0.35) or eGFR ( β =0.10, 95% CI, −0.07 to 0.28). Conversely, CKD associated with a diminished risk of kidney stones in multivariable MR models (UKBB/FinnGen: OR=0.77, 95% CI, 0.69 to 0.87; FinnGen/UKBB: OR=0.73, 95% CI, 0.66 to 0.81). Furthermore, in the Mayo Clinic cohort with available urinary biochemistries, lower eGFR was associated with lower urinary calcium excretion and urinary calcium oxalate/phosphate supersaturation. Conclusions In this study, kidney stones were not independently associated with CKD. Conversely, CKD was associated with a lower risk of calcium kidney stones likely via changes in key urinary traits, including lower calcium excretion.

The primary cilium is a microtubule-based sensory organelle that plays a critical role in signaling pathways and cell cycle progression. Defects in the structure and/or function of the primary cilium result in developmental diseases collectively known as ciliopathies. However, the constituents and regulatory mechanisms of the primary cilium are not fully understood. In recent years, the activity of the epigenetic modifier SMYD3 has been shown to play a key role in the regulation of cell cycle progression. However, whether SMYD3, a histone/lysine methyltransferase, contributes to the regulation of ciliogenesis remains unknown. Here, we report that SMYD3 drives ciliogenesis via the direct and indirect regulation of cilia-associated components. We show that SMYD3 is a novel component of the distal appendage and is required for centriolar appendage assembly. The loss of SMYD3 decreased the percentage of ciliated cells and resulted in the formation of stumpy cilia. We demonstrated that SMYD3 modulated the recruitment of centrosome proteins (Cep164, Fbf1, Ninein, Ttbk2 and Cp110) and the trafficking of intraflagellar transport proteins (Ift54 and Ift140) important for cilia formation and maintenance, respectively. In addition, we showed that SMYD3 regulated the transcription of cilia genes and bound to the promoter regions of C2cd3, Cep164, Ttbk2, Dync2h1 and Cp110. This study provides insights into the role of SMYD3 in cilia biology and suggests that SMYD3-mediated cilia formation/function may be relevant for cilia-dependent signaling in ciliopathies.