Hongquan Geng

Primary hyperoxaluria type 1 (PH1) is a rare inherited liver disorder caused by alanine glyoxylate aminotransferase (AGT) dysfunction, leading to accumulation of glyoxylate which is then converted into oxalate. Excessive oxalate results in kidney damage due to deposition of oxalate crystals. We have developed an mRNA-based protein replacement therapy for PH1 to restore normal glyoxylate to glycine metabolism. Sequence optimized human AGT mRNA ( hAGT mRNA) was encapsulated in lipopolyplex (LPP) and produced functional AGT enzyme in peroxisomes. Pharmacokinetics and pharmacodynamics (PK/PD) were evaluated in vitro and in vivo. PK demonstrated that AGT mRNA and AGT protein maintained high expression levels for up to 48 hours. A single 2 mg/kg dose in Agxt Q84 ^−/− rats achieved a 70% reduction in urinary oxalate. Toxicological assessment identified the highest nonserious toxic dose (HNSTD) as 2 mg/kg. These findings affirm the efficacy and safety of hAGT mRNA/LPP and support its clinical application in PH1 treatment.

Genome-wide association studies (GWAS) have identified loci associated with kidney diseases, but the causal variants, genes, and pathways involved remain elusive. Here, we identified a kidney disease gene called pancreatic progenitor cell differentiation and proliferation factor (PPDPF) through integrating GWAS on kidney function and multiomic analysis. PPDPF was predominantly expressed in healthy proximal tubules of human and mouse kidneys via single-cell analysis. Further investigations revealed that PPDPF functioned as a thiol-disulfide oxidoreductase to maintain cellular NAD ⁺ levels. Deficiency in PPDPF disrupted NAD ⁺ and mitochondrial homeostasis by impairing the activities of nicotinamide mononucleotide adenylyl transferases (NMNATs), thereby compromising the function of proximal tubules during injuries. Consequently, knockout of PPDPF notably accelerated the progression of chronic kidney disease (CKD) in mouse models induced by aging, chemical exposure, and obstruction. These findings strongly support targeting PPDPF as a potential therapy for kidney fibrosis, offering possibilities for future CKD interventions.

Purpose To compare the clinical characteristics of pediatric urolithiasis patients with positive and negative molecular diagnoses. Methods The clinical characteristics corresponding to pediatric urolithiasis patients that had undergone exome sequencing at our hospital between January 2016 and May 2021 were collected. Genetic analysis results were used to separate patients into positive and negative molecular diagnosis groups. Multivariate logistic regression analyses adjusted for visiting age, sex, ethnicity, province, and body mass index were used to compare differences in medical history, diagnostic imaging findings, and renal function between individuals with and without molecular diagnoses. Results In total, 194 patients with pediatric urolithiasis of unknown etiology underwent exome sequencing and were included in the present study, of whom 63 obtained urolithiasis-related molecular diagnoses. Relative to cases without a molecular diagnosis, those with a positive molecular diagnosis were more likely to be associated with a positive family history (OR 2.84, 95% CI 1.29–6.29, p = 0.008), consanguineous parents (OR 24.7, 95% CI 1.34–454, p = 0.002), early onset (OR 1.26, 95% CI 1.09–1.45, p < 0.001), nephrocalcinosis (OR 10.6, 95% CI 3.06–36.6, p < 0.001), cast stone (OR 18.9, 95% CI 4.40–81.1, p < 0.001), multiple stones (OR 13.9, 95% CI 6.39–30.2, p < 0.001), bilateral stones (OR 7.04, 95% CI 3.47–14.2, p < 0.001), a lower estimated glomerular filtration rate (OR 1.17, 95% CI 1.07–1.28, p < 0.001), and chronic kidney disease (OR 26.9, 95% CI 1.42–526, p < 0.001). Conclusion A positive family history, consanguineous parents, early onset, nephrocalcinosis, severe stone burden, and impaired renal function are signals of concern that are suggestive of inherited urolithiasis.

Background: Primary hyperoxaluria type 1 (PH1) is a rare genetic disorder caused by endogenous overproduction of hepatic oxalate, leading to hyperoxaluria, recurrent calcium oxalate kidney stones, and end‐stage renal disease. Lactate dehydrogenase (LDH) is an ideal target for diminishing oxalate production as it is responsible for glyoxylate to oxalate conversion in the liver, the last step of oxalate metabolism. Here, we investigated the therapeutic efficacy and potential side effects of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology to ameliorate PH1 via specifically disrupting the hepatic LDH.Methods: Pheochromocytoma (PC12) cells were used to assess the efficacy of cleavage of single‐guide RNAs in vitro. PH1 neonatal rats were injected with a single administration of adeno‐associated virus to deliver the CRISPR/Cas9 system that targeted LDH. Three weeks after injection, a liver biopsy was performed to detect LDH expression, liver injury, and liver metabolomics. Urinary oxalate was regularly monitored, and renal calcium oxalate deposition was evaluated after 4 weeks of 0.5% ethylene glycol challenge. After 6 months of treatment, animals were euthanized, and ex‐liver organs were harvested for toxicity analysis.Results: The Ldha gene was specifically knocked out in 20% of the liver cells of PH1 rats in the treatment group, leading to a 50% lower LDH expression than that in the control group. Compared to the control groups, urinary oxalate levels were significantly decreased, and renal calcium oxalate precipitation was largely mitigated in the treatment group throughout the entire 6‐month study period. While no CRISPR/Cas9‐associated off‐target edits or hepatotoxicity were detected, we observed mild metabolic changes in the liver tricarboxylic acid (TCA) and glycolysis pathways.Conclusions: CRISPR/Cas9‐mediated LDH disruption may represent an applicable new strategy for alleviating PH1 for its long‐lasting effect and low editorial efficiency requirements.

Purpose: Ureteropelvic junction obstruction (UPJO) is the most frequent cause of congenital hydronephrosis in child. To better investigate the molecular mechanisms of this pathological process, the stenotic ureter proteome of UPJO in infants is compared with their own normal pre‐stenotic segments.Experimental Design: Data independent acquisition‐based proteomics are performed to compare proteome between pre‐stenotic and stenotic ureter from nine UPJO infants. Gene ontology analysis, hierarchical cluster analysis, and network interaction are performed to characterize biological functions of significantly altered proteins. Selected significantly altered proteins are validated by western blot on another three UPJO infants.Results: 15 proteins are up‐regulated and 33 proteins are down‐regulated during stenotic pathology. Significantly altered proteins are involved in decreased extracellular matrix and cytoskeleton organization, increased regulation of oxidative activity, and altered inflammatory associated exocytosis. Significant expression of biglycan, fibulin‐1, myosin‐10, cytochrome b5 are validated providing possible mechanism in UPJO which could be associated impaired smooth muscle cell, epithelial integrity, and increased oxidative stress.Conclusions and Clinical Relevance: This study provides molecular evidence of dysregulated extracellular matrix organization, impaired smooth muscle cell, and oxidative stress during UPJO pathology, indicating that biglycan, fibulin‐1, myosin‐10, cytochrome b5 might reflect the pathology of UPJO.

Purpose To investigate the prevalence of inherited causes in an early onset urolithiasis cohort and each metabolic subgroup. Methods A retrospective analysis of both metabolic and genomic data was performed for the first 105 pediatric urolithiasis patients who underwent exome sequencing at our hospital from February 2016 to October 2018. Measurements included the diagnostic yield of exome sequencing in the entire cohort and each metabolic subgroup (hyperoxaluria, hypocitraturia, hypercalciuria, hyperuricosuria and cystine stone subgroups). The conformity between molecular diagnoses and metabolic evaluation was also evaluated. Results The present study involved a cohort of 105 pediatric patients with urolithiasis, from which diagnostic variants were identified in 38 patients (36%), including 27 primary hyperoxaluria and 11 cystinuria. In the metabolic subgroup analyses, 41% hyperoxaluria cases were primary hyperoxaluria caused by monogenic defects, and 100% of the causes of cystine stones could be explained by monogenic defects. However, no appropriate inherited causes were identified for hypocitraturia, hypercalciuria, or hyperuricosuria in the cohort. A high conformity (100%) was obtained between the molecular diagnoses and metabolic evaluation. Conclusion Exome sequencing in a cohort of 105 pediatric patients with urolithiasis yielded a genetic diagnosis in 36% of cases and the molecular diagnostic yield varies substantially across different metabolic abnormalities.